Genetic changes in atm and atr/chek1 as prognostic indicators in cancer

ABSTRACT

The present invention relates to the discovery that, in human cancer, an 11q deletion of ATM together with an increase in ATR and CHEK1 expression correlates with resistance to ionizing radiation which could be overcome by inhibition of the ATR/CHEK1 pathway. It provides for methods of identifying patients unlikely to exhibit an adequate response to radiation therapy and/or chemotherapy who may benefit from ATR/CHEK1 pathway inhibition, as well as methods of treating said patients.

PRIORITY CLAIM

This application is a continuation of U.S. Ser. No. 13/407,165, filedFeb. 28, 2012, which is a divisional application of U.S. Ser. No.12/586,052, now U.S. Pat. No. 8,173,366, filed on Sep. 15, 2009, whichis a continuation-in-part of U.S. patent application Ser. No.12/079,900, filed Mar. 28, 2008, now U.S. Pat. No. 8,263,329, and claimspriority to U.S. Provisional Application No. 60/908,891, filed Mar. 29,2007; U.S. Provisional Application No. 60/912,086, filed Apr. 16, 2007;and U.S. Provisional Application No. 60/912,355, filed Apr. 17, 2007,the contents of each of which are incorporated by reference in theirentireties herein.

GRANT INFORMATION

The subject matter of the present invention was developed, at least inpart, under National Institutes of Health Grant No. RO1DE14729,P30CA47904, RO1DE016086, R25CA089507 and P50CA097190, so that the UnitedStates Government has certain rights herein.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herein via EFS on Apr. 23, 2013. Pursuant to 37 CFR1.52(e)(5), the Sequence Listing text file, identified asSequencelisting.txt, is 6,799 bytes and was created on Apr. 23, 2013.The Sequence Listing does not extend beyond the scope of thespecification and thus does not contain new matter.

1. INTRODUCTION

The present invention relates to the discovery that, in human cancer, an11q deletion of ATM together with an increase in ATR and CHEK1expression correlates with resistance to ionizing radiation which couldbe overcome by inhibition of the ATR/CHEK1 pathway. It provides formethods of identifying patients unlikely to exhibit an adequate responseto radiation therapy and/or chemotherapy who may benefit from ATR/CHEK1pathway inhibition, as well as methods of treating said patients.

2. BACKGROUND OF THE INVENTION 2.1 Oral Squamous Cell Carcinoma

Worldwide, head and neck squamous cell carcinoma (HNSCC) is the sixthmost common cancer; it is the third most common cancer in developingnations. There were 404,575 new cases in 2002, constituting 3.7% of thetotal estimated cancer cases worldwide (PARKIN et al., 2002). Accordingto a recent study by the American Cancer Society, in 2006, there wereexpected to be approximately 30,990 new cases of HNSCC and 7,430 deathsrelated to HNSCC (JEMAL et al. 2006).

HNSCC encompass tumors of the tongue, oral cavity, pharynx and larynx.Of these, tumors of the oral cavity and tongue, that is oral squamouscell carcinomas (OSCC) account for more than 90% of all HNSCC(SCHANTZand YU 2002). Although the elderly population accounts for most of thecases of OSCC, recently there has been a significant increase in OSCCincidence in the younger age group (MARTIN-GRANIZO et al. 1997,SILVERMAN 2001).

Leukoplakia, erythroplakia, nicotine stomatitis, tobacco pouch keratosisand oral submucosal fibrosis are common premalignant lesions which mayprogress to OSCC (NEVILLE and DAY 2002). Leukoplakia, which is a whitediscoloration in the lining of the oral cavity, is very common in theelderly population. Nearly 10-15% of leukoplakia lesions progress toOSCC over a period of time (PETTI 2003).

The common risk factors for oral squamous cell carcinoma are tobaccochewing, smoking, alcohol consumption and human papillomavirus (HPV)infection (RHODUS 2005). Vitamins A and C, on account of theirantioxidant activity, are shown to be protective against the developmentof oral leukoplakia and squamous cell carcinoma (NAGAO et al. 2000). Thecombined effects of tobacco use, alcohol consumption and poor dietaryhabits account for over 90% of head and neck cancer cases (REICHART2001).

In addition to tobacco and alcohol consumption, dietary habits, geneticpredisposition, and unknown risk factors may play a role in theincreased incidence of OSCC observed recently (LLEWELLYN et al. 2004).In spite of better methods of diagnosis and new procedures fortreatment, there has not been a significant difference in the five yearsurvival rate for HNSCC over the past 30 years; it was 59% during1995-2000 and 54% during 1974-1976 (JEMAL et al. 2006). Thus, there is aneed to develop new biomarkers for effective screening, and therapeuticstrategies for treatment of leukoplakia and OSCC.

2.2 Chromosomal Instability

One hallmark of solid tumors is chromosomal instability, which helpsdrive cancer growth and progression. As the tumor progresses, itsgenotype evolves into one that is optimized for proliferation, spreadand invasion into surrounding tissues. Thus, over a period of time,genetic alterations that confer a growth advantage are selected(ALBERTSON et al. 2003). Chromosomal instability is a state ofcontinuous, dynamic, propagated changes in chromosome structure and/ornumber, which is usually seen in solid tumors.

OSCC exhibits a high level of chromosomal instability with near-triploidor tetraploid karyotypes composed of multiple clonal numerical andstructural chromosomal abnormalities (VAN DYKE et al. 1994, GOLLIN 2001,JIN et al. 2002). Chromosomal segregational defects, involvingmultipolar spindles are a common cause of numerical chromosomalinstability in OSCC (SAUNDERS et al. 2000, GISSELSSON et al. 2002). Theanaphase bridges and micronuclei observed in OSCC may be caused by DNAdefects in the damage response and/or dysfunctional telomeres (GOLLIN2001, GISSELSSON 2003). Anaphase bridges are intermediates in theprocess of gene amplification (SHUSTER et al. 2000, SAUNDERS et al.2000).

Changes in DNA ploidy and chromosomal abnormalities can be detected intumor-adjacent normal tissue and dysplastic tissue and also inpremalignant lesions of the oral cavity (SUDBO et al. 2002). Thus,chromosomal instability is a relatively early event in head and necktumor development. A high degree of chromosomal aneuploidy and changesin DNA ploidy in OSCC are correlated with lymph node metastasis, poorresponse to treatment and poor prognosis (HOLM 1982, STELL 1991). Eventhough OSCC exhibits a high degree of cytogenetic heterogeneity, certainchromosomal changes like 11q13 amplification, gains involving 3q21-29,5p, 8q, 18q and 22q and losses involving 3p, 8p, 9p, 11q, 13q and 21qoccur regularly and may play a role in OSCC development and progression(BOCKMUHL and PETERSEN 2002).

Amplification of chromosomal band 11q13, one form of chromosomalinstability, is present in nearly 45% of OSCC (GOLLIN 2001). 11q13amplification is an independent prognostic factor which correlates withhigher stage disease, lymph node involvement, shorter time torecurrence, and reduced overall survival (AKERVALL et al. 1997,FRACCHIOLLA et al. 1997, MICHALIDES et al. 1997). Gene amplification,the generation of extra copies of a gene or genes, is a common geneticdefect in human tumors, including OSCC. Amplification and subsequentoverexpression of critical genes has been shown to lead to dysregulationof the cell cycle, resulting in cellular proliferation and tumorformation and/or progression (LUNDBERG et al. 1999).

Chromosomal band 11q13, which harbors the locus for a key cell cycleregulatory gene, cyclin D1 gene (CCND1) and other neighboring genes, isthe most frequently amplified genetic segment in OSCC (AKERVALL et al.1997, GOLLIN 2001, HUANG et al. 2002). 11q13 amplification is alsopresent in a smaller percentage of other carcinomas (SCHRAML et al.1999). Hittelman and colleagues report that 11q13 amplification occursearly in the pathogenesis of OSCC, in premalignant lesions prior todevelopment of invasive carcinoma (IZZO et al. 1998). 11q13amplification with cyclin D1 overexpression is a critical event in thepathogenesis of OSCC and is associated with a poor prognosis (AKERVALLet al. 1997).

11q13 amplification in OSCC occurs by the breakage-fusion-bridge (BFB)mechanism, first described in maize by geneticist Barbara McClintock in1938. The first step in the BFB model of 11q13 amplification is loss ofa distal portion of chromosome 11, resulting in an unprotectedchromosome end which fuses with its sister chromatid to form a dicentricchromosome. During chromosomal segregation, the two centromeres arepulled to different poles, leading to additional breaks and thus thecycle continues till a derivative chromosome 11 with 11q13 amplificationis formed (SHUSTER et al. 2000). During the initial step of 11q13amplification, a segment of distal 11q harboring important genesinvolved in DNA damage recognition and repair like MRE11A (11q21), ATM(11q22.3), H2AFX (11q23.2) and CHEK1 (11q24) are lost.

2.3 ATR and CHEK1

CHEK1 and CHEK2 (also referred to as CHK1 and CHK2) are highly conservedeffector kinases which transmit signals from ATM and ATR to downstreamproteins. This in turn leads to cell cycle checkpoint activation, andDNA repair (BARTEK and LUCAS 2003). CHEK1 mainly acts during the G2 andS phases of the cell cycle to initiate cell cycle checkpoints inresponse to DNA damage (LIU et al. 2000). The early lethality ofCHEK1-deficient embryonic cells (TAKAI et al. 2000) and early embryoniclethality of CHEK1-deficient mice (LIU et al. 2000) suggests that CHEK1plays a very important role during early development and viability inmammals (KALOGEROPOULOS et al. 2004).

CHEK1 induced G2M arrest is mediated through phosphorylation andinactivation of the Cdc25 phosphatases (SANCHEZ et al. 1997). Inaddition to CHEK1 and CHEK2, a number of other proteins like p53, MDM2,c-ABL, SMC1, BRCA1, BRCA2, and the Fanconi proteins are phosphorylatedand activated by ATM and ATR (SHILOH et al. 2001a, SHILOH et al. 2001b).Thus, perturbation of either ATM or ATR may have a ripple effect andaffect the regulation and control of numerous proteins and pathwaysinvolved in the DNA damage response.

The aim of the G2 (G2M) checkpoint is to prevent cells with improper DNAreplication or DNA damage from entering mitosis. The critical regulatorof the G2 checkpoint is the pro-mitotic cyclin B/CDK 1 kinase complexwhich in turn is regulated by the ATR, CHEK1 and CDC25 phosphatasefamily of proteins (O'CONNELL et al. 2005). The G2 checkpoint partlyrelies on p53-dependent mechanisms. However, it has been shown thatp53-independent mechanisms are sufficient to sustain G2 arrest. Entry ofcells with DNA damage into mitosis results in activation of an Mphase-specific, p53-independent pathway that results in mitoticcatastrophe and apoptosis (O'CONNELL et al. 2005). It has been shownthat knockout of either CHEK1 or ATR results in mitotic catastropheduring embryogenesis or loss of replication checkpoint control incycling Xenopus extracts (HEKMAT-NEJAD et al. 2000).

Since the G1 phase checkpoint is lost in a large number of tumors, thereis growing interest in S and G2 phase checkpoint abrogating agents whichcan sensitize these tumors to chemotherapy and radiotherapy (ZHOU et al.2003).

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery that, in human cancer, an11q deletion of ATM together with an increase in ATR and CHEK1expression correlated with resistance to ionizing radiation which couldbe overcome by inhibition of the ATR/CHEK1 pathway. It is based, atleast in part, on the discovery that both ATR and CHEK1 areoverexpressed in a subset of oral squamous cell carcinomas (“OSCCs”)with loss of the G1 cell cycle checkpoint, and that non-specificinhibition of ATR or CHEK1 with caffeine or specific inhibition with therespective siRNAs resulted in an increased susceptibility of the OSCCsto DNA damaging agents. It is further based, in part, on the results ofFISH evaluation of a variety of cancer cell lines which demonstrated thepresence of 11q loss as well as amplification of cyclin D1 (CCND1), agene in the chromosomal band 11q13 locus, in a substantial percentage ofthe cancers tested. It is still further based on the discovery thatdecreased or absent p53 expression and/or activity is associated withresistance to ionizing radiation and susceptibility to inhibition of theCHEK1 pathway, so that p53 is another biomarker that may be usedaccording to the invention.

In one set of non-limiting embodiments, the present invention providesfor methods of identifying patients unlikely to exhibit an adequateresponse to radiation therapy who may benefit from ATR/CHEK1 pathwayinhibition, as well as methods of treating said patients. For example,such a patient may be identified by detecting, in a cancer cell of saidpatient, a loss of 11q together with an increase in the expression ofATM and/or CHEK1, optionally an increase in CCND1 (which may occur withloss of 11q), and optionally a decrease or absence of p53 expressionand/or activity.

In further non-limiting embodiments, the present invention provides forkits which may be used to identify patients who may be resistant toradiation therapy and/or may benefit from ATR/CHEK1 pathway inhibition.

4. BRIEF DESCRIPTION OF THE FIGURES COLOR FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. QuMA studies to map segmental loss of microsatellite loci ondistal 11q. QuMA was performed for each of the OSCC cell lines in thethree groups. The cell lines were grouped as (A) “no 11q13amplification, no distal loss”, (B) “11q13 amplification with distal 11qloss” and (C) “distal 11q loss but no 11q13 amplification”.

FIG. 2. Loss of heterozygosity (“LOH”) studies performed on OSCC formultiple loci on distal 11q. LOH studies for multiple markers onchromosome 11q demonstrate loss of distal loci or genes in cell linesfrom each of the three groups: A “11q13 amplification with distal 11qloss”, B “distal 11q loss but no 11q13 amplification” and C “no 11q13amplification, no distal loss”.

FIG. 3. RNA expression changes for MRE11A, ATM, H2AX and CHEK1 in OSCC.Qualitative Real-Time PCR (“qRT-PCR”) was performed for the four genesMRE11A, ATM, H2AX and CHEK1 on distal 11q. Overall genetic loss of ATMand H2AX correlated well with a reduced expression. UPCI:SCC084, 136,142 and 125 show increased CHEK1 expression in spite of loss at the genelevel. UP3_(—)344, 348 and 700 are normal human oral keratinocytes(control cell lines). R=red; O/R=orange red (predominantly red); R/O=redorange; O=orange; Y/O=yellow orange; O/Y=orange yellow; Y=yellow;G/Y=green yellow; Y/G=yellow green; G=green; G/DG=green dark green;DG=dark green.

FIG. 4. Protein expression changes for MRE11A, ATM and H2AX in OSCC.Immunoblotting was performed to detect changes in protein expression forMRE11A, ATM and H2AX. We observed reduction in MRE11A, ATM and H2AXprotein in most cell lines with loss irrespective of their amplificationstatus.

FIG. 5A-B. γ-H2AX focus formation at the end of 1 h following 2.5 Gy IRtreatment. The distribution of γ-H2AX focus formation was evaluated in(A) untreated OSCC cell lines, NHOK and an AT cell line (GM09607) and(B) OSCC cell lines, NHOK and an AT cell line treated with 2.5 Gy IR atthe end of 1 h.

FIG. 6. Clonogenic cell survival of OSCC to IR compared with controlNHOK. Grouped analysis for clonogenic cell survival demonstrates thatthe cell lines with no amplification but with loss (red), and cell lineswith amplification and loss (blue) show a similar survival, while celllines with no amplification and no loss (orange) show survivalcomparable to the control NHOK. At a very high IR dose of 10 Gy, celllines with 11q loss irrespective of their amplification status have anaverage of 9% cells surviving.

FIG. 7A-D. Comprehensive summary of DNA damage-related results inrepresentative cell lines, showing results from the FISH, focusformation and chromosome aberration assays. UPCI:SCC116 has noamplification of 11q13 or loss of distal 11q. UPCI:SCC084 has 11q13amplification and loss of distal 11q. UPCI:SCC104 has loss of distal11q, but no 11q13 amplification. In (A), FISH images showing copy numberin each of the cell lines. In each FISH image, CEP 11 is labeled red(indicated by an arrow pointing at the image) and H2AFX is labeled green(indicated by a circle around the image). UPCI:SCC116, which isnear-triploid, shows the expected complement of three red and threegreen signals. UPCI:SCC084, which is near-diploid, has two CEP 11signals and only one green signal. UPCI:SCC104, which is near-triploid,has four copies of the chromosome 11 centromere and only two copies ofH2AFX. In (B), γ-H2AX focus formation in control and treated (2.5 Gy IRwith 1 h repair) cells. After treatment, UPCI:SCC116 shows a largenumber of foci, indicating recognition of double-strand breaks.UPCI:SCC084 and UPCI:SCC104 have higher baseline levels of focusformation, and markedly fewer foci in response to equivalent doses ofradiation. All focus formation images are composite images of five 0.2μm z-stacks. Examples of common breakage events are shown for each cellline in (C), as indicated with a green arrow. The metaphase spread forUPCI:SCC116 does not show any breaks two days post-IR, but UPCI:SCC084and UPCI:SCC104 show numerous breakage events at the same time point. Asummary of the total weighted aberrations per cell for representativecell lines are shown in (D). A chromosomal breakage assay was carriedout on three cell lines, one with “No Amplification, No Loss”(UPCI:SCC116), another with “Loss, No Amplification” (UPCI:SCC104) and athird with “Amplification with Loss” (UPCI:SCC084). Control, white; IR,gray. Chromosome aberrations are weighted as previously described, andthen normalized to a diploid cell. In brief, two-break events includingchromosome breaks, radials, giants, rings and dicentrics were assignedtwice the weight of other aberrations. The total weighted aberrationswere summed, and determined per chromosome and per cell for eachtreatment. Error bars are provided as the standard error of the samplemean for both control and treated samples.

FIG. 8. Comparison of cell cycle profiles of UPCI:SCC066 and 104 inresponse to IR. UPCI:SCC066 and 104 were either mock treated or treatedwith 5 Gy IR and allowed to recover for 24 h. At the end of 24 h, flowcytometric analysis show complete loss of the G1 cell cycle checkpointin UPCI:SCC104 with most cells accumulating in the S and G2M phases,UPCI:SCC066 cells accumulate at both G1 and G2M cell cycle checkpoints.

FIG. 9. p53 and ATR expression in a subset of OSCC. Loss of p53expression correlates with an increased expression of ATR protein inUPCI:SCC084, 104, 131, 131, 136 and 142. UPCI:SCC103, which has atruncated p53 protein also has ATR overexpression. Expression inUPCI:SCC066 and 172 is comparable to that observed in HEK 293 cells.

FIG. 10A-F. Structural and numerical changes in ATR and CHEK1 genes intumor cell lines. (A) Demonstrates two copies of ATR gene (green) andtwo CEP 3 (red) signals in normal metaphase. (B) We observed atranslocation of the ATR gene (green) in UPCI:SCC084. In UPCI:SCC104 (C)ATR gene (green) is gained with isochromosome 3q formation. In anovarian tumor cell line, OVCAR-3 (D), we observe amplification of theATR gene (green) compared to CEP 3 (red). (E) Represents normalmetaphase with two CHEK1 (red) and two CEP 11 (green) signals (F)UPCI:SCC131 with loss of the CHEK1 gene (red) compared to CEP 11(green).

FIG. 11A-F. Results for ATR, CHEK1 and CCND1 FISH in primary head andneck tumors and adjacent normal tissue. (A-B) demonstrate gain in ATRgene (green) copy number compared with CEP 3 (red) in normal (A) andtumor (B); (C-D) show loss of CHEK1 gene (red) compared to CEP 11(green) in normal (C) and tumor (D); (E-F) demonstrate amplification ofCCND1 gene (red) compared to CEP 11 (green) in tumor tissue but not inthe adjacent normal tissue.

FIG. 12A-D. ATR translocation in SCC084 and isochromosome 3 formation inSCC104. (A) shows translocation of one copy of ATR gene (green) fromchromosome 3 (red), further analysis of UPCI:SCC084; (B) reveals thatATR (green) is translocated to the derivative chromosome 11 (red) withamplified CCND1 (aqua); (C) demonstrates UPCI:SCC104 with anisodicentric chromosome 3 and ATR gain (green); (D) is a magnificationof the isodicentric chromosome shown in (C).

FIG. 13A-B. Presence of ATR (green), CEP11 (aqua) and CEP3 (red) inanaphase bridge. UPCI:SCC104 (Panel A) demonstrates two ATR copies andtwo CEP 3 copies in the anaphase bridge. In GM09607 (Panel B), two ATRcopies and two CEP 3 copies are present in the anaphase bridge (whitearrow) while one CEP 11 copy is present in another anaphase bridge (redcolor).

FIG. 14A-C. qRT-PCR analysis for ATR and CHEK1 RNA expression in subsetof OSCC. (A) demonstrates heat map of ATR (top row) and CHEK1 (bottomrow) expression, (B) Bar graph depicting relative ATR expression (C) Bargraph depicting relative CHEK1 expression.

FIG. 15. ATR and CHEK1 protein expression in OSCC detected byimmunoblotting. Immunoblotting for ATR and CHEK1 demonstrates high ATRand CHEK1 expression in UPCI:SCC084, 104, 131, 136, 142 and 172.

FIG. 16A-C. Activation of downstream effectors of ATR in response to IRand UV. (A) Phosphorylation of CHEK1 on serine-345 was studied inresponse to 5 Gy IR and 20 J/m2 dose of UV radiation in UPCI:SCC066,084, 104 and 105. (B) Phosphorylation of SMC1 on serine-957 was studiedin response to 20 J/m2 dose of UV radiation in UPCI:SCC066, 084, 104 and105. (C) Phosphorylation of Cdc25C serine-216 was studied in response to5 Gy IR UPCI:SCC066 and 104.

FIG. 17. Cell cycle profiles of UPCI:SCC066 and 104 in response to IRand caffeine treatment. UPCI:SCC066 (Panel A) and UPCI:SCC104 (Panel B)were either treated with 5 Gy IR or pretreated with 1 mM caffeine 1 hprior to treatment with 5 Gy IR, or pretreated with 5 mM caffeine 1 hprior to 5 Gy IR and the cell cycle profiles were compared withuntreated samples from the same cell line. Compared to SCC066, SCC104exhibits a loss of G1 checkpoint and predominant G2M accumulationfollowing treatment with IR. SCC104 exhibits a caffeine-dose dependentreduction in G2M accumulation and increase in sub G0 peak (dead cells)suggesting that caffeine sensitizes SCC104 to IR mediated cell death.

FIG. 18A-C. The frequency of PCC formation (cell death) in untreatedcells, in response to aphidicolin with or without 1 mM caffeinepretreatment. (A) Demonstrates the frequency of mitotic cells undergoingPCC/MC in OSCC, NHOK and GM09607. (B,C) Depicts PCC/MC formation inUPCI:SCC104 under the specified conditions.

FIG. 19. Clonogenic cell survival of UPCI:SCC084 to different doses ofcaffeine. Complete inhibition of colony formation in UPCI:SCC084 at adose of 1 mM caffeine.

FIG. 20. Clonogenic cell survival of OSCC to different doses ofcaffeine. UPCI:SCC084 and 104 exhibit increased sensitivity to caffeinecompared to UPCI:SCC066, 105 and control NHOK.

FIG. 21. ATR and CHEK1 siRNA mediated protein knockout in UPCI:SCC104.We observed nearly complete loss of ATR and CHEK1 protein expression atthe end of 72 h following treatment with ATR and CHEK1 siRNAsrespectively.

FIG. 22A-B. Flow cytometric analysis following treatment of ATR andCHEK1 siRNA. Cell cycle profiles of (A) UPCI:SCC104 and (B) UPCI:SCC066following treatment with non-specific siRNA, ATR siRNA and CHEK1 siRNAin non irradiated cells or cells irradiated with 5 Gy IR are depicted.In comparison to SCC066, SCC104 shows increased accumulation ofirradiated cells in the G2M phase. On inhibition of ATR or CHEK1 withthe respective siRNAs we observe elimination of the G2M accumulation ofirradiated cells and an increase in the sub-G0 dead cell population.

FIG. 23. Induction of premature chromatin condensation and mitoticcatastrophe following ATR and CHEK1 siRNA treatment. Frequency of PCC/MCin UPCI:SCC066 and SCC:104 in response to ATR and CHEK1 siRNA treatmentwith or without aphidicolin (Aph). The numbers next to the barsrepresent the percentage of mitotic cells undergoing PCC/MC. Bar 1=CHK1siRNA+Aph; bar 2=CHK1 siRNA; bar 3=ATR siRNA+Aph; bar 4=ATR siRNA; bar5=non-specific siRNA+Aph; bar 6=non-specific siRNA; bar 7=Aph; and bar8=untreated.

FIG. 24A-B. Clonogenic cell survival of UPCI:SCC066 and 104 to ATR siRNAtreatment. UPCI:SCC066 (Panel A) and UPCI:SCC104 (Panel B) cells at 50%confluence were mock treated with empty Lipofectamine or treated with anon-specific scrambled siRNA or treated with ATR siRNA and cell survivalwas compared to untreated tumor cells for each cell line. A modestreduction in cell survival was observed in UPCI:SCC066, whileUPCI:SCC104 cells were highly sensitive to ATR inhibition.

FIG. 25. Important genes that can be amplified or lost on distal 11q.

FIG. 26. LOH profiles for a subset of the HNSCC cell lines groupedaccording to 11q status: “Amplification with Loss” (UPCI:SCC078, 084,131 and 136), “Loss, No Amplification” (UPCI:SCC104 and 142), “NoAmplification, No Loss” (UPCI:SCC099 and 116) and UPCI:SCC125(heterogeneous, with some tendency toward “distal 11q loss without 11q13amplification”). The approximate locations of CCND1 in addition to theDNA damage response genes are indicated along the left side of thefigure. Also provided, are the map distances from the centromere (in M)and the microsatellite loci or genes that were analyzed for loss ofheterozygosity. A key is provided below the figure to indicate the LOHstatus of each locus in the cell lines. Allelic loss greater than 90% ata given locus was called “LOH,” allelic loss greater than 50% was called“partial LOH,” and allelic loss less than 50% was considered “no LOH.”An “X” indicates an allele mismatch at that particular locus.

FIG. 27. Phosphorylation of H2AX 1 h after exposure to 2.5 Gy IR. Celllines are clustered according to 11q status as described previously,with the addition of a normal fibroblast cell line as a positive controland GM09607 as a negative control. The levels of γ-H2AX are shown inmock-treated (C) and after 1 h of repair (IR). Results are derived froma single observer-generated dataset and two independent datasetsgenerated by a spot-counting algorithm on the MetaSystems Metaferscanning system (MetaSystems, Altlussheim, Germany), and displayed asthe mean level of focus formation (±95% CI). The mean levels of focusformation after treatment are generally lower in HNSCC cells with lossof distal 11q, irrespective of 11q13 amplification.

FIG. 28A-B. Results from the clonogenic survival assay. In (A), thesurviving fraction of cells at each dose of ionizing radiation isplotted with error bars (±SD) for the HNSCC cell lines sorted by groups.Surviving fraction is plotted on a logarithmic scale. Note that therewere no surviving cells remaining for the “No Amplification, No Loss”cell lines or the NHOK cells. HNSCC cells with distal 11q loss showsurvival at 10 Gy IR, irrespective of 11q13 amplification status.UPCI:SCC125 is seen to have low level survival relative to the celllines with loss of distal 11q. Individual survival curves (±SD) for thethree cell lines are shown in (B). The surviving fraction is plotted ona logarithmic scale. Note that there were no surviving UPCI:SCC116 (“NoAmplification, No Loss”) cells after 10 Gy IR. In contrast, the two celllines with distal 11q loss had ˜10% of cells surviving 10 Gy IR,irrespective of 11q13 amplification.

FIG. 29A-B. Expression assays for mRNA and protein. The qRT-PCRexpression profiles for the DNA damage response genes assayed in theHNSCC cell lines in (A) Three cell lines with “No Amplification, NoLoss,” four HNSCC cell lines with “Amplification with Loss,” and threecell lines with “No Amplification with Loss.” UPCI:SCC125 is includedadjacent to the third group, and three NHOK cell lines are provided ascontrols. A key is provided on the left, indicating the relativefold-changes in expression. (B) The immunoblot data are shown forMRE11A, ATM and H2AX expression in the HNSCC cell lines. Also includedis a representation of γ-H2AX induction following 2.5 Gy of IR. MRE11Aand ATM were normalized to α-actinin, and H2AX was normalized to α-actinto control for errors in loading prior to a densitometric analysis. Theimages provided for total protein expression are a composite of twogels, and the images provided for the γ-H2AX expression are a compositeof four gels. All protein gels have been truncated to show the band ofinterest.

FIG. 30A-B. Stacked column distribution of foci in mock-treated andtreated (2.5 Gy) cells. These data are derived from a single experiment,and are intended to complement the mean number of foci per cell. Thedistribution (0-2, 3-10 and >10 foci) is represented as a percentagecontribution to the total number of cells scored in each cell line. Inpreliminary experiments, 95% of the untreated NHOK had 2 or fewer fociper cell. Therefore, 0-2 foci is the baseline grouping. In (a), themock-treated populations of cells show primarily 0-2 foci, with someexceptions (UPCI:SCC099 and 131 show slightly high numbers of foci atbaseline). In (b), cells treated with 2.5 Gy IR show shifts toward 3-10or more foci in the population, and fewer cells with 0-2 foci. Thisshift is most prominent in the normal fibroblasts and “No Amplification,No Loss” HNSCC cell lines as well as UPCI:SCC125. In contrast, theGM09607 cells show little change in focus distribution in mock-treatedand treated populations.

FIG. 31. Results from a viability assay in representative cell lines.Cellular viability was assessed under the same conditions as γ-H2AXimmunofluorescence, in order to predict what fraction of cells with fociwere not undergoing repair (apoptotic). This viability assay is based onthe integrity of the cell membrane. Results for the assay carried out intriplicate are presented as 95% confidence intervals, and indicate thatthere is equivalent survival in treated and mock-treated populations.Based on these findings, any cell death seen in 1 h is not attributableto the effect of treatment. The implication is that the small numbers ofnon viable cells encountered during the focus formation assay did notinfluence the sample mean or distribution of foci representative of DNAdouble strand breaks.

FIG. 32A-B. Western blots showing p53 status of (a) oral squamouscarcinoma and (b) non-small cell lung carcinoma and ovarian carcinomacell lines as assessed by the presence of p53 and p21 proteins after a4-hour treatment with adriamycin.

FIG. 33. Clonogenic cell survival of ES-2 (without active p53) ovariancarcinoma cells treated with 2.5 Gy irradiation versus non-irradiatedcontrol.

FIG. 34. Clonogenic survival of AS49 (with active p53) cells treatedwith 2.5 Gy irradiation versus non-irradiated control.

FIG. 35. Clonogenic survival of UPCI:SCC040 (without active p53) cellseither non-irradiated or irradiated with either 2.5 or 5 Gy of radiationand either untreated (control) or treated with siRNA to CHEK1.

5. DETAILED DESCRIPTION OF THE INVENTION

For clarity of description, and not by way of limitation, the presentinvention is divided into the following subsections:

(i) methods of identifying 11q deletion;

(ii) methods of identifying ATR/CHEK1/CCND1/p53 copy number alterationsand/or overexpression;

(iii) methods of identifying target patients;

(iv) methods of treating target patients; and

(v) kits.

5.1 Methods of Identifying 11q Deletion

In one non-limiting aspect of the invention, the present inventionprovides for identifying an 11q deletion in a patient sample.

The term “11q deletion” refers to a deletion in the long arm of humanchromosome 11 which comprises at least a part of the ATM gene and mayfurther comprise at least a portion of MRE11A, H2AFX, and/or CHEK1.

A patient sample is a sample that contains genomic material. Forexample, but not by way of limitation, the sample may be a cell, atissue, or DNA. In a specific, non-limiting embodiment, the samplecomprises a cancer cell.

The deletion in 11q may be identified by any method known in the art. Innon-limiting embodiments, it may be detected using a nucleic acid proberepresenting a gene or non-coding sequence located on 11q, preferablyATM, MRE11A, H2AFX, CHEK1, genes located at 11q21-23 such as, but notlimited to, BACE, BLR1, CBL, DDX10, DFNB24, DLAT, DNCH2, FRA11H, FSGS2,G6PT1, HEPHL1, HSPB2, etc. (a list of genes located on 11q may be foundat (http://www.gdb.org/gdbreports/GeneByChromosome.11.alpha.html orhttp://genome.ucsc.edu) or in other genome databases known the skilledartisan. Sequences from this region preferably lacking a repeatedelement may also be used. In non-limiting embodiments, single nucleotidepolymorphism (SNP) analysis may be used to show copy number loss. Thefollowing Table 1 sets forth genes which may be lost with an 11qdeletion.

TABLE 1 Distance from Gene name Location centromere Function PAK111q13.5 76.8 Mb P21 activating kinase regulates cell motility andmorphology FAT3 11q14.3 91.9 Mb Tumor suppressor homo log of DrosophilaFAT JRKL 11q21 95.8 Mb Nuclear factor DDI1 11q22.3 103.4 Mb Induced inresponse to DNA damage CASP1, 4, 5 11q22.1-22.3 104.3 Mb Effectors ofapoptosis INCA 11q22.3 104.5 Mb Regulates recruitment and activation ofprocaspase 1 ICEBERG 11q22.3 104.5 Mb Caspase 1 inhibitor NPAT 11q22.3107.5 Mb Nuclear PPP2R1B 11q23 110.8 Mb Protein phosphatase regulatorysubunit REXO2 11q23.1 113.2 Mb 3′-5′ exonuclease involved in DNA repairUSP28 11q23.1 113.3 Mb De-ubiquitinating enzyme ZBTB16 11q23.1 113.4 MbZinc finger transcription factor regulates histone deacetylase and cellcycle progression UBEA 11q23.3 118 Mb Conjugation factor required forpoly- and multi ubiquitination POU2F3 11q23.3 119.6 Mb Induces terminalkeratinocyte differentiation BRCC2 11q24.1 121.5 Mb BH3-like domaincontaining pro-apoptotic protein

In non-limiting embodiments of the invention, loss of 11q may correlatewith an increase in expression in cyclin D1 (CCND1), so that an increasein expression of CCND1 may be used as an indicator of loss of 11q (anincrease in CCND1 expression is consistent with loss of 11q, althoughloss of 11q does not necessarily result in an increase in expression ofCCND1). Further genes which may be lost or amplified are shown in FIG.25.

The nucleic acid probe may be hybridized to genomic DNA in the form of achromosome (e.g., by fluorescence in situ hybridization (“FISH”)).Quantitative Microsatellite Analysis (QuMA) may be used to detect copynumber changes in microsatellite markers along chromosome arm 11q. PCRprimers sequences for the microsatellite loci are shown in Appendix D.In a specific non-limiting example, (see Section 7), the TaqManCA-repeat fluorogenic probe 5′-FAM (6-carboxyfluorescein)-TGTGTGTGTGTGTGTGTGTGT-TAMRA(6-carboxytetramethylrhodamine)-3′ (SEQ ID NO:26) may be used(Integrated DNA Technologies, Coralville, Iowa). Other methods wouldinclude quantitative DNA PCR, loss of heterozygosity (LOH) analysis, orarray comparative genomic hybridization (“CGH”) to identify 11q deletionand gene loss. In each case, it is preferable to include, in the study,a control probe, such as a chromosome 11 centromere probe.

In alternative embodiments, 11q deletion may be identified by karyotypeanalysis, preferably with attention to the 11q22 region where the ATMgene resides.

In a preferred, non-limiting embodiment, as exemplified in section 6below, dual-color FISH may be performed along with BAC probes to one ormore of MRE11A, ATM, H2AFX and CHEK1 along with a centromere 11enumeration probe (CEP 11; D11Z1) (Vysis/Abbott Molecular Inc., DesPlaines, Ill.). This working example provides non-limiting examples ofprobes which may be used. (See also Appendix A).

5.2 Methods of Identifying ATR/CHEK1/CCND1 Copy Number Alterationsand/or Overexpression

In non-limiting aspects of the invention, the present invention providesfor identifying overexpression of ATR and/or CHEK1 and optionally CCND1and/or p53 (and/or p53 activity) in a patient sample.

Any method known in the art may be used to measure expression of ATRand/or CHEK1 and optionally CCND1 and/or p53.

As one non-limiting example, the location and copy number of ATR and/orCHEK1 and optionally CCND1 and/or p53 may be determined using FISH.

In a specific non-limiting example, ATR may be present in amplified formon chromosome 3 (again, preferably a control probe, such as a chromosome3 centromere probe, may be included as a control).

As one non-limiting example, quantitative real-time or reversetranscriptase PCR (“qRT-PCR”) may be used to measure expression of ATRand/or CHEK1 and optionally CCND1 and/or p53.

As another non-limiting example, a Northern blot may be used to measureexpression of ATR and/or CHEK1 and optionally CCND1 and/or p53.

As another non-limiting example, the level of ATR and/or CHEK1 andoptionally CCND1 protein and/or p53 may be measured using a method knownin the art (e.g. a Western blot or immunohistochemically).

For example, but not by way of limitation, the sequence of ATR may beaccessed as Genbank Acc. No. NM_(—)001184; the sequence of CHEK1 may beaccessed as Genbank Acc. No. NM_(—)001274; and the sequence of CCND1 maybe accessed as Genbank Acc. No. NM_(—)053056.

“Overexpression” as that term is used herein means an increase of atleast about two-fold or at least about five-fold, or preferably at leastabout ten-fold in the RNA or protein level for a particular gene,compared to expression in normal control cells.

Specific non-limiting examples of nucleic acid probes (defined toinclude hybridization probes as well as primers) may be found in Section6 and Appendices A and B, below. Specific non-limiting examples ofantibodies which may be used to measure protein levels are presented inAppendix C.

Further, the presence of active p53 may be evaluated by determiningwhether or not a gene downstream of p53 in an induction pathway isinduced. One non-limiting example of such a gene is p21 (Lee et al.,200, Proc. Natl. Acad. Sci. U.S.A. 97:8302-8305, incorporated byreference). Induction of a gene may be evaluated using a method ofdetermining protein expression as known in the art, including thosemethods described above.

5.3. Methods of Identifying Target Patients

A “target patient”, as that term is used herein, is a patient who fallswithin a subset of patients having a cancer which is more likely to beresistant or refractory to radiation (or other free radical based)therapy or chemotherapy and/or which may benefit from a treatment whichincludes inhibition of the ATR/CHEK1 pathway.

A “target patient” may suffer from any cancer. In preferred non-limitingembodiments of the invention, the cancer is a squamous cell carcinoma,more preferably a squamous cell carcinoma of the head or neck region(“HNSCC”), most preferably oral squamous cell carcinoma (“OSCC”). Othercancers associated with ATM copy number change or mutation which maybenefit from the present invention include, but are not limited to,melanoma, ovarian cancer, lung cancer, kidney cancer, leukemia,lymphoma, multiple myeloma, pancreatic cancer, prostate cancer, coloncancer, esophageal cancer, cervical cancer, and breast cancer.

Accordingly, the present invention provides for a method of identifyinga patient suffering from a cancer which is likely to be refractory totherapy based on ionizing radiation or other free-radical generatingmethod and/or chemotherapy, and/or which is likely to benefit from (i.e.be treatable by) inhibition of the ATR/CHEK1 pathway, comprising (i)identifying an 11q deletion in a patient sample considered to berepresentative of the cancer (e.g., a tumor sample, or a tumor cell, ora malignant cell not associated with a solid tumor, or a DNA sample fromone of the foregoing, etc.) and/or (ii) identifying, in the patientsample, overexpression of ATR and/or CHEK1 and/or CCND1 and optionally(iii) determining whether p53 expression and/or activity is decreased orabsent, wherein an 11q deletion and/or overexpression of ATR and/orCHEK1 and/or CCND1 and/or a decrease in expression and/or activity ofp53 indicates that the cancer is likely to be resistant or refractory totherapy based on ionizing radiation or other free-radical generatingmethod and/or is likely to benefit from inhibition of the ATR/CHEK1pathway.

In a related embodiment, the present invention provides for a method,which may be a further step in the method described in the precedingparagraph, of identifying a patient suffering from a cancer which islikely to be refractory to therapy based on ionizing radiation or otherfree-radical generating method and/or chemotherapy, and/or which islikely to benefit from inhibition of the ATR/CHEK1 pathway, comprising(i) exposing cells in a patient sample considered to be representativeof the cancer to a source of free-radicals such as ionizing radiation;and (ii) determining whether there is a higher percentage of cells inthe S and G2M phases relative to control cells (representative of normaltissue); where a higher percentage of cells in the S and G2M phasesindicates that the cancer is likely to be refractory to therapy based onionizing radiation or other free-radical generating method and/orchemotherapy and/or is likely to benefit from inhibition of theATR/CHEK1 pathway.

5.4 Methods of Treating Target Patients

The present invention provides for a method of treating a target patientin need of such treatment with an effective amount of an inhibitor ofthe ATR/CHEK1 pathway as part of a treatment regimen that includes atleast one DNA-damaging agent. In preferred non-limiting embodiments, thetarget patient has been identified as likely to benefit from suchtherapy using a method described in the preceding section.

Inhibitors of the ATR/CHEK1 pathway include inhibitors whichspecifically inhibit the expression and/or function of ATR and/or CHEK1.Non-limiting examples include siRNA, antisense, and catalytic RNAs whichshare homology with the ATR or CHEK1 genes, including mixtures thereof.For example, but not by way of limitation, the sequence of ATR may beaccessed as Genbank Acc. No. NM_(—)001184; and the sequence of CHEK1 maybe accessed as Genbank Acc. No. NM_(—)001274. These RNA inhibitors maybe, in non-limiting embodiments, nucleic acids between about 15 and 100bp, or between about 15 and 50 bp, or between 15 and 30 bp in length,and may contain deoxyribonucleosides or non-natural bases. Smallmolecule inhibitors, such as chemical inhibitors of the protein,receptor, or specific monoclonal antibodies may be used. Small moleculeinhibitors include, but are not limited to CHIR-124, XL844, A-690002,A-641397 and A-901592 (see, e.g., Chen, et al., 2006, Intl. J. Cancer119:2784-2794). Said inhibitor(s) may be administered by any suitableroute, including, but not limited to, intravenous, intra-arterial,intrathecal, intra-peritoneal, subcutaneous, intranasal, pulmonaryinhalation, and direct instillation into the tumor or tumor bedfollowing complete or partial surgical resection.

DNA-damaging agents which may be used according to the inventioninclude, but are not limited to, ionizing radiation, either appliedexternally or using a radioactive implant, or free radical generators,for example, but not limited to, chemotherapeutic agents (e.g., thatbind to DNA) and ultraviolet radiation.

The DNA-damaging agent may be administered to the patient prior to,concurrently with, or subsequent to, administration of the inhibitor ofATR/CHEK1, however, it is desirable to select the timing ofadministration of both agents so that the effect of the DNA-damagingagent is not suppressed by overexpression of ATR and/or CHEK1 and/oractivation of the ATR/CHEK1 pathway. Preferably, but not by way oflimitation, the DNA-damaging agent is administered within 24 hours ofinhibiting ATR and/or CHEK1.

ATR/CHEK1 inhibitor together with DNA-damaging agent, used according tothe invention, may be administered as part of a treatment regimen whichincludes concurrent or sequential treatment with one or more otheranti-cancer agent, including but not limited to conventionalchemotherapeutic agents and cytokines, such as cisplatinum, paclitaxel,5-fluorodeoxyuridine, and/or carboplatinum.

In non-limiting embodiments, the invention provides for a method oftreating a patient suffering from a cancer, comprising (i) identifyingthe patient as being likely to benefit from inhibition of the ATR/CHEK1pathway by a method comprising (a) identifying an 11q deletion in apatient sample considered to be representative of the cancer and/or (b)identifying, in the patient sample, overexpression of a gene selectedfrom the group consisting of ATR, CHEK1, CCND1 and a combinationthereof, wherein an 11q deletion and overexpression of ATR, CHEK1, CCND1or a combination thereof, indicates that the cancer is likely to benefitfrom inhibition of the ATR/CHEK1 pathway; and (ii) treating the patientwith an inhibitor of the ATR/CHEK1 pathway.

In further non-limiting embodiments, the present invention provides fora method of treating a patient suffering from a cancer, comprising (i)identifying the patient as being likely to benefit from inhibition ofthe ATR/CHEK1 pathway by a method comprising (a) identifying an 11qdeletion in a patient sample considered to be representative of thecancer and/or (b) identifying, in the patient sample, overexpression ofa gene selected from the group consisting of ATR, CHEK1, CCND1 and acombination thereof, and (c) identifying, in the patient sample, adecrease in p53 expression and/or activity wherein an 11q deletion andoverexpression of ATR, CHEK1, CCND1 or a combination thereof and/or adecrease in p53 expression and/or activity indicates that the cancer islikely to benefit from inhibition of the ATR/CHEK1 pathway; and (ii)treating the patient with an inhibitor of the ATR/CHEK1 pathway.

5.5 Kits

The present invention further provides for kits that comprise elementsthat may be used to identify a deletion in 11q as well as overexpressionof ATR and/or CHEK1 and optionally CCND1. Such a kit may comprisenucleic acid probes for identifying and/or directly or indirectlydetecting an ATM gene and one more gene selected from the groupconsisting of ATR, CHEK1, CCND1, MRE11A and H2AFX.

A “nucleic acid probe” as that term is used herein includes but is notlimited probes suitable for hybridization (e.g., for FISH) as well asprimers (e.g., for PCR or QRT-PCR); said nucleic acids may be betweenabout 15 and 200, or between about 20 and 100, or between about 15 and50 nucleotides in length or larger (e.g., 100-200 kb for a FISH probe),and capable of hybridizing to the gene of interest, for example underconditions suitable for FISH or QRT-PCR.

A kit may further optionally comprise one or more antibody directedtoward ATM, ATR, CHEK1, MRE11A, H2AFX, CCND1 or CHEK2 protein with orwithout phosphorylation.

A nucleic acid probe or antibody contained in such a kit may itself bedetectably labeled or may be indirectly detectable via a detectablemolecule capable of binding to said probe or antibody.

A detectable label provides a signal which may be, for example, but notbe way of limitation, fluorescent, radioactive, pigmented, etc.

6. EXAMPLE The DNA Damage Response Pathway in Oral Squamous CellCarcinoma

Oral squamous cell carcinomas are a good model system for analyzinggenetic alterations relating to chromosome 11, since nearly 45% of OSCCare characterized by amplification of chromosomal band 11q13, whichoccurs by a breakage-fusion-bridge (“BFB”) cycle mechanism. The firststep in the BFB cycle involves breakage and loss of distal 11q.Consequently, numerous genes, including critical genes involved in theDNA damage response pathway (e.g., MRE11A, ATM, and H2AFX) are lost inthe step preceding 11q13 amplification.

The experiments described herein were designed, in part, to evaluate theeffect of loss of genes on distal 11q on the DNA damage response inOSCC, which is representative of other cancers which are associated withdistal 11q loss. Characterization of OSCC using FISH revealed partialloss of MRE11A, ATM, and H2AFX in all cell lines with 11q13amplification and in addition lines lacking this amplification.Quantitative microsatellite analysis and loss of heterozygosity studiesconfirmed the distal 11q loss. Reverse transcriptase quantitative PCRand immunoblotting revealed reductions in RNA and protein expression ofMRE11A, ATM and H2AFX that correlated with genetic loss. All cell lineswith distal 11q loss exhibited a decrease in the size and number ofγ-H2AX foci and increased chromosomal instability following treatmentwith ionizing radiation. Surprisingly, distal 11q loss was alsocorrelated with reduced sensitivity to ionizing radiation. Although theliterature attributes the poor prognosis in OSCC and other cancers to11q13 amplification, the results presented herein indicate that distal11q deletions may be equally if not more significant.

Further, the ATR/CHEK1 pathway was found to be upregulated in a subsetof OSCC. The experiments described herein were performed, in part, todetermine whether the upregulated ATR/CHEK1 pathway protects OSCC frompremature chromatin condensation or mitotic catastrophe (leading to celldeath) by enhancing the S phase and G2 phase checkpoints, and toevaluate whether inhibiting this pathway would sensitize OSCC to DNAdamaging agents.

As described below, a gain in ATR gene copy number, but a partial lossof CHEK1 at the gene level, was observed in OSCC. However, in a subsetof OSCC cells with loss of the G1 cell cycle checkpoint, overexpressionof both ATR and CHEK1 was observed. Nonspecific inhibition of ATR orCHEK1 with caffeine or specific inhibition with the respective siRNAswas found to result in increased susceptibility of OSCC to DNA damagingagents, including ionizing radiation.

6.1 Materials and Methods

Subjects and Sample Collection.

OSCC cell lines were established from tumors surgically removed fromanonymous consenting, previously untreated patients. Normal humankeratinocytes (NHOK) were established from uvulopalatopharyngeal tissueobtained from University of Pittsburgh Medical Center. The tissue wascollected from anonymous consenting patients with IRB approval.Peripheral blood for FISH was collected from normal anonymous donors.GM09607, AT fibroblast cell line was purchased from Coriell CellRepositories. The OKF6/TERT-1 cell line was obtained from Dr. JimRheinwald.

Cell Culture.

Eleven OSCC cell lines were selected for the distal 11q loss study andtwenty OSCC cell lines were selected for ATR and CHEK1 studies from theOSCC established in the laboratory. The ATM-deficient, AT cell line(GM09607) was used as a positive control since it is documented to havean upregulated ATR-CHEK1 pathway, and normal human oral keratinocytes(NHOK), OKF6/TERT-1 and/or HEK293 cell line were used as negativecontrols.

OSCC Cell Lines.

OSCC were cultured in M10 medium composed of Minimal Essential Medium(Gibco Invitrogen, Grand Island, N.Y.), supplemented with 1%non-essential amino acids, 1% L-glutamine, 0.05 mg/ml gentamicin and 10%fetal bovine serum (FBS) (Gibco Invitrogen). For subculturing OSCC,adherent cells were detached from the flask surface by trypsinizing with0.05% trypsin and 0.02% EDTA (Irvine Scientific) for 3-5 min at 37° C.in 5% CO2 incubator. Equal amounts of M10 medium were used to inhibittrypsin activity following detachment and cells were replated.

AT Null Cell Line.

GM09607 (Coriell Cell Repositories, Camden, N.J.) was cultured usingDulbecco's Modified Eagle Medium (DMEM) (Gibco Invitrogen), supplementedwith 1% non-essential amino acids, 0.05 mg/mlpenicillin-streptomycin-L-glutamine, and 10% FBS. Subculturing wasperformed as described for OSCC.

Controls.

Anonymous NHOK cells established from uvulopalatopharyngoplastyspecimens were used as controls. In brief, NHOK cells were cultured inserum-free KGM-2 medium (Clonetics, Walkersville, Md.), supplementedwith bovine pituitary extract (BPE), hEGF, insulin (bovine),hydrocortisone, GA-1000 (Gentamicin, Amphotericin B), epinephrine andtransferrin as per the manufacturer's instructions (supplements suppliedin the KGM-2 BulletKit™, Clonetics). The hTERT cells were cultured inKeratinocyte-SFM supplemented with 25 μg/ml BPE, 0.2 ng/ml epidermalgrowth factor, 0.3 mM CaCl2 and penicillin-streptomycin (GibcoInvitrogen). These keratinocytes were expanded to high density in a 1:1mixture of Keratinocyte-SFM and DMEM-F12. The DMEM-F12 was a 1:1 mixtureof calcium-free and glutamine-free DMEM and Ham's F-12 supplemented with25 μg/ml bovine pituitary extract, 0.2 ng/ml epidermal growth factor,1.5 mM L-glutamine and penicillin-streptomycin (Gibco Invitrogen).

Preparation of DNA Probes for FISH

A single colony of E. coli carrying the individual BAC (mapping to ATR,ATM, CHEK1, CCND1 H2AFX, MRE11A and TP53) (Individual BAC numbers arelisted in Appendix A) was incubated overnight at 37° C. in 5 ml ofLuria-Bertani (LB) medium with 50 μg/ml Chloramphenicol. The bacteriawere centrifuged at 10,000×g for 30 s. The bacteria were resuspended in100 μl of STET (8% sucrose, 5% Triton X100, 50 mM EDTA, 50 mM Tris pH8.0). Freshly prepared alkaline SDS (0.2 NaOH, 1% SDS) was added to lysethe bacteria and the solution was incubated at 24° C. for 2 min. Coldammonium acetate (4° C.) was added and the solution was incubated for 5min on ice. Following this step, the bacteria were centrifuged at 4° C.for 15 min at 16,000×g. Equal amounts of phenol and chloroform wereadded to the supernatant to extract the DNA. The top layer of themixture was treated with 0.6× volume of isopropanol and centrifuged at4° C. for 15 min at 16,000×g. The supernatant was drained and the pelletwashed with 70% ethyl alcohol and air dried. The DNA was resuspended in100-200 μl of Tris-EDTA (TE) buffer (QIAGEN, Valencia, Calif.) andstored at 4° C.

Fluorescence In Situ Hybridization (FISH).

In order to prepare mitotic cells for FISH analyses, HNSCC cells wereharvested following 5 h of 0.1 μg/ml Colcemid™ (Irvine Scientific, SantaAna, Calif.) treatment, hypotonic KCl (0.075M) treatment for 16 min andfixation in 3:1 methanol:glacial acetic acid. All other cells wereharvested using the same method, except that 1 h of Colcemid™ waspreferred for non-tumor cells. FISH analysis was used to detect copynumber changes for the respective genes in the OSCC cell lines. For FISHanalysis, cells were harvested, dropped onto slides, treated withRNase/2×SSC, and dehydrated using serial treatments with 70%, 80% and100% of ethyl alcohol. Chromatin was denatured with 70% formamide anddehydrated in 70%, 80% and 100% of ethyl alcohol. The BAC probes forFISH, described in detail in Appendix A, were obtained from Children'sHospital of Oakland Research Institute (CHORI, San Francisco, Calif.).Using a nick translation kit from Vysis, Inc. (Downers Grove, Ill.),extracted DNA was precipitated with ethyl alcohol, resuspended inhybridization buffer, and allowed to pre-anneal for 1-2 h at 37° C. Eachprobe was hybridized for 16 h at 37° C., after which slides were washedwith SSC/Tween-20. Slides were counterstained with DAPI and mounted withantifade prior to analysis. All FISH analyses were carried out using anOlympus BX-51 epifluorescence microscope (Olympus Microscopes, Melville,N.Y.). An Applied Imaging CytoVision workstation with Genus v3.6software was used for image capture and analysis (Applied Imaging, SanJose, Calif.).

Paraffin FISH.

A 4-5 μM thick slice of paraffin embedded tissue was mounted on apositively charged microscope slide. The slides were aged overnight at60° C., following which they were deparaffinized with Xylene for 5 minat room temperature. The slides were dehydrated with a series of 70%,80% and 100% ethyl alcohol washes, each wash lasting 2 min at roomtemperature. The slides were then treated with 0.5×SKIP Dewax solutionat 80° C. for 15 min followed by treatment with pepsin containingprotease solution for 15 min at 37° C. After two washes with 2×SSC, eachlasting 5 min, the slides were fixed in 10% Formalin for 10 min at roomtemperature. After 2×SSC washes, the slides were dehydrated with aseries of 70%, 80% and 100% ethyl alcohol washes and allowed to dry onslide warmer. The probes for paraffin FISH were prepared as describedfor regular FISH. The hybridization of the FISH probes and posthybridization treatment of the paraffin slides was carried out asdescribed for regular FISH. Unless specified otherwise, 100 nuclei fromtumor tissue and 100 nuclei from normal tissue were analyzed for copynumber changes of different genes.

Anaphase Bridge Formation Assay.

To check for presence of ATR gene in anaphase bridges, each OSCC cellline was plated in chamber slides and allowed to grow until the celllines reached 80-90% confluence. 5 ml of Colcemid™ was added to eachchamber slide and the slides were incubated at 37° C. in 5% CO2incubator for a period of 24 h. At the end of 24 h, the media wasaspirated and cells were fixed with 3:1 methanol to acetic acid fixativefor a period of 45 min. FISH using BAC probe to the ATR gene labeledwith Spectrum Green™ CEP3 labeled with Spectrum Orange™ and CEP11labeled with Spectrum Aqua™ was performed as described above. Fiftyanaphase bridges per OSCC or GM09607 were evaluated for the presence ofATR, CEP3 or CEP11.

Clonogenic Cell Survival Assay.

To assess cell survival in response to ionizing radiation, clonogenicsurvival assays were performed. Two thousand cells were seeded in 60 mmPetri dishes and allowed to adhere overnight. Cells were then treatedwith increasing doses of γ-irradiation at 1, 2.5, 5, and 10 Gy usingGammacell 1000 Elite irradiator (Nordion International, Inc., Ottawa,Canada) with a 137Cs source at a dose rate of 4.42 Gy/min. The culturemedium was replaced at the end of 7 days. Untreated cells cultured inparallel were used to determine relative plating efficiency. After 12days, the cells were fixed with 70% ethyl alcohol and stained withGiemsa (Sigma, St. Louis, Mo.) and the number of colonies was assessed.A colony was defined as a cluster of ≧50 cells, having formed from asingle cell. All experiments were performed in triplicate, and the errorreported as one standard deviation from the mean.

RNA Extraction and Real-Time PCR.

RNA extraction for real time PCR was performed using TRIzol reagent(Invitrogen) according to the manufacturer's instructions. The extractedRNA was purified using the RNeasy Mini kit (QIAGEN) and resuspended in100 μl RNase free water. The RNA samples were purified of unwanted DNAwith the help of DNA-free DNase kit (Ambion, Austin, Tex.) according tothe manufacturer's instructions. RNA concentrations were determinedusing the SmartSpec 3000 (Bio-Rad Laboratories) and normalized to 40ng/μl. Reverse transcription was carried out with three inputs for eachsample: 400 ng of total RNA, 100 ng of total RNA and a negative controlwith no reverse transcriptase. The RT set up is described in Table 2below:

TABLE 2 QRT-PCR reagents Input No reverse Reagent Company 400 ng 100 ngtranscriptase 10 x PCR Buffer II Applied Biosystems 10 μl 10 μl 10 μlMgCl₂ (25 Mm) Applied Biosystems 30 μl 30 μl 30 μl dNTP (25 μM) RocheMolecular Biochemicals 4 μl 4 μl 4 μl MMLV 10 U/μl Ambion 1 μl 1 μl 0 μlRNase Inhibitor (40 U/μl) Applied Biosystems 1 μl 1 μl 1 μl Hex Primer(500 μM) Applied Biosystems 2.5 μl 2.5 μl 2.5 μl Nuclease free WaterAmbion 41.5 μl 49 μl 42.5 μl RNA (amount) 10 μl 2.5 μl 10 μlThe thermocycler conditions were set up as: 25° C. for 10 min, 48° C.for 40 min, 95° C. for 5 min and hold at 10° C. The cDNA was diluted 2.5times to yield working concentrations of 1.6 ng/μl and 0.4 ng/μl.

For quantitative PCR (qRT-PCR), 5 μM of each primer, 10 μM of probe, 25mM dNTPs, 25 mM MgCl₂, AmpliTaq Gold enzyme (Applied Biosystems) wereused. The Taqman primers and probes for ATR, CHEK1 and the control, 18SrRNA were obtained from Applied Biosystems. qRT-PCR was carried out at95° C. for 10 min followed by 40 cycles of 95° C. for 15 s and 60° C.for 60 s using the 7300 Real-Time PCR System (Applied Biosystems). Eachsample was run in triplicate along with the no reverse transcriptasecontrol. For each plate, at least three wells were set up with themaster mix but without any cDNA template (no template control). The RNAexpression levels were quantified relative to the Universal ReferencecDNA obtained from Clontech (Mountain view, Calif.).

Immunoblotting.

Immunoblotting was utilized to detect protein expression of MRE11A, ATMand H2AX in HNSCC cell lines, and also to assess the phosphorylationlevels of H2AX following exposure to 2.5 Gy IR. Flasks of each cell linewere trypsinized, washed with ice cold 1× phosphate-buffered saline(PBS) and lysed on ice with a solution containing 50 mM Tris, 1% TritonX-100 (Sigma), 0.1% sodium dodecyl sulfate (Bio-Rad Laboratories,Hercules, Calif.), 150 mM NaCl (Fisher Chemicals, Fairlawn, N.J.), 1 mMdithiothreitol (DTT) (Fisher Scientific, Inc., Hampton, N.H.), 10 μg/mlleupeptin (Roche Applied Science, Indianapolis, Ind.), 10 μg/mlpepstatin (Roche Applied Science), and 1 nM phenyl methyl sulfonylfluoride (PMSF) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.).The soluble cell lysate was centrifuged at 2000×g for 15 min andtransferred to a clean microcentrifuge tube. Histone H2AX was extractedfrom the remaining pellet containing insoluble protein and chromatin.The pellet was treated with 0.1% HCl for 20 min on ice, and thencentrifuged at 10,000×g for 10 min. The supernatant was againtransferred to a clean microfuge tube.

Protein concentrations resulting from the standard and acid lysisprocedures were determined using the Bio-Rad Quick Start BradfordProtein Assay Kit using a SmartSpec 3000 (Bio-Rad Laboratories). Theacid lysate was neutralized with Tris-EDTA (TE) pH 8.0 prior tonormalization. Normalized lysates with a protein concentration of 1μg/μl were resolved by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto an Immobilon-P membrane(Millipore Corporation, Billerica, Mass.). After blocking with 5%non-fat dry milk (NFDM) for 1 h, the membrane was incubated overnightwith the desired primary antibody (Appendix C) at room temperature.Following three 5 min washes in 1×TBST (Tris-buffered Saline Tween-20),the membrane was incubated with the appropriate secondary antibody(1:3000) for 2 h. Target proteins were visualized using the WesternLighting™ Chemiluminescence Reagent Plus kit (PerkinElmer Life Sciences,Boston, Mass.) according to the manufacturer's instructions.

To verify equal protein loading in the gels, membranes were stripped andre-probed with antibodies against β-tubulin (Santa Cruz), actin (SigmaImmunochemicals, St. Louis, Mo.). Individual antibodies, theirconcentrations and characteristics are listed in Appendix C.

Cell Cycle Analysis by Flow Cytometry.

Following the relevant treatments, mock, IR or aphidicolin, and with orwithout caffeine or respective siRNAs, floating and adherent cells werecollected at the end of 24 h. These cells were washed withphosphate-buffered saline (PBS), and fixed with 70% ethanol. The cellswere then treated with 80 μg/ml RNase A and 50 μg/ml propidium iodide(Invitrogen-Molecular Probes, Carlsbad, Calif.) for 45 min at 37° C. Thestained cells were analyzed using a Coulter Epics XL Flow Cytometer inthe UPCI Flow Cytometry Facility.

Chromosome Breakage Studies.

To check for chromosomal damage in OSCC in response to ionizingradiation, the total weighted aberrations per cell were determined forUPCI:SCC084, 104 and 116. Briefly, UPCI:SCC084, 104 and 116 weresubjected to 2.5 Gy of IR. The cells were re-seeded and allowed torepair for 48 h prior to being harvested after treatment with 18 nMCalyculin A for 30 min (Calbiochem, San Diego, Calif.). Harvested cellswere subjected to a hypotonic treatment before being fixed in Carnoy'sfixative. Slides were then prepared from the cell pellets, solid stainedfor 8 min in 4% Giemsa/PBS solution, and rinsed with distilled water. 50cells per case (case includes control and IR-treated cells) were scored.Chromosome aberrations documented include: chromosome breaks, chromatidgaps or breaks, radials, giants, rings, minutes, dicentrics, fragmentsand dots. Chromosome breaks, radials, giants, rings and dicentrics wereassigned twice the weight of the other aberrations since they involvetwo chromatid events. The total weighted aberrations were summed, anddetermined per chromosome and per cell for each treatment. The standarderror of the mean was used as the estimate of error in the sample. AStudent's t-test was used to compare the raw distributions of totalweighted aberrations between the control and IR-treated samples.

PCC Induction in OSCC.

To determine whether inhibition of the ATR-CHEK1 pathway can sensitizeOSCC to DNA damaging agents, we treated OSCC cell lines with aphidicolin(Sigma), a DNA polymerase-inhibitor to induce DNA damage, and caffeine(Sigma), a nonspecific kinase inhibitor to inhibit ATR activity. We usedAT cell line (GM09607) with a deficient G1 phase checkpoint as ourpositive control and NHOK cells as negative controls. Briefly 75-80%confluent flasks of cells were pretreated with 1 mM caffeine for 30 minfollowing which 0.4 μM aphidicolin was added. The cell lines wereharvested for metaphases 24 h after aphidicolin treatment. Afluorescence microscope was used to count mitotic cells that hadcharacteristic features of either normal mitosis or PCC. Criteria fordistinguishing PCC from normal metaphase were adapted from a previousreport (NGHEIM et al. 2001). Briefly, interphase cells and cells thatwith intermediate morphology between normal and PCC were not included inthe analysis. Partial metaphases with PCC were also not included in theanalysis. The following criteria were used to identify mitoses as PCC ornormal: PCC characteristics include well-defined particles by DAPIstaining that were round, not oblong, particles with no hazy chromatinmaterial; no chromatid-like pairs present; and borders of the cell'schromatin were irregular and composed of speckles, not smooth or with ahazy appearance (all characteristics must be met). Characteristics ofnormal mitoses include well-defined chromosomes with a primaryconstriction; at least 40 such chromosomes should be found in eachmetaphase spread.

SiRNA Transfection.

ATR or CHEK1 inhibitions were carried out using the respective siRNAsfor a specific knockout. RNA interference of ATR and CHEK1 was performedusing Smartpool ATR and CHEK1 duplexes respectively, obtained fromDharmacon (Lafayette, Colo.). Nonspecific (scrambled) control duplexes(Dharmacon) were used for nonspecific siRNA treatment. The duplexes werereconstituted in 1×DNA-free RNA re-suspension buffer provided byDharmacon and aliquoted according to the manufacturer's instructions.For transfection, the OSCC cell lines were seeded in 60 mm dishes or T25flasks and transfected with siRNA duplexes using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. Theindividual siRNA duplex sequences are enumerated in Appendix B. Thefinal working siRNA concentration achieved was between 90-100 nM. Weused cells treated with no vector (untreated), empty vector(mock-treated), cells transfected with a nonspecific siRNA, and aspecific ATR or CHEK1 siRNA for all of our experiments. At the end of 48h post-transfection, appropriate treatments (ionizing radiation oraphidicolin treatment) were carried out as described earlier.

6.2 Results

A Segment of Distal 11q is Partially Lost in a Subset of OSCC.

According to the BFB model, the first step in 11q13 amplification isloss of a segment of distal 11q. To determine whether genes located ondistal 11q and involved in the DNA damage response are lost in OSCC, wecarried out dual-color FISH with BAC probes to MRE11A, ATM, H2AFX andCHEK1 along with a centromere 11 enumeration probe (CEP 11; D11Z1)(Vysis, Downers Grove, Ill.). Table 3 summarizes our FISH results interms of copy number loss in relation to the ploidy of each cell line,derived from chromosome 11 centromere enumeration and consensuskaryotypes. The copy number ratios of the distal 11q genes were normalor lost with respect to CEP 11. The eleven cell lines were grouped basedon FISH assessment of 11q13 amplification and distal 11q loss asfollows: most cell lines with 11q13 amplification demonstrated partialloss of copies of all four genes, including UPCI:SCC078, 084, 131 and136 (“11q13 amplified with distal loss”). The OSCC cell lines,UPCI:SCC104, 142 and 122 had loss of one or more genes in the absence of11q13 amplification (“Distal 11q loss but no 11q13 amplification”).UPCI:SCC099, 116 and 182 did not have 11q13 amplification or distal 11qloss (“no 11q13 amplification, no distal loss”). Interestingly,UPCI:SCC078 and 104 did not demonstrate loss of the CHEK1 gene.UPCI:SCC125 is comprised of a highly heterogeneous cell population,making its analysis less straightforward than the other cell lines.There is no evidence of 11q13 amplification in this cell line. While thecopy number ratios seem to show that UPCI:SCC125 is relatively normal incopy number for each of the four distal genes, FISH results indicatedthat results for MRE11A (31% loss, 44% gain), ATM (52% loss, 1% gain),H2AFX (43% loss, 25% gain) and CHEK1 (45% loss, 30% gain) are more afunction of the average than the norm.

TABLE 3 Summary of FISH copy number changes for MRE11A ATM, H2AFX andCHEK1 in OSCC cell lines.

¹Shading indicates partial loss or haploinsufficiency. ²UPCI:SCC125 is ahighly heterogeneous cell line with respect to copy number showingcomparable number of cells with gain, loss or normal copy number for anygiven gene.Confirmation of physical loss on distal 11q was carried out usingquantitative microsatellite analysis or QuMA. QuMA was used to map theDNA copy number of segmental microsatellite loci along 11q (FIG. 1). Themicrosatellites, D11S1358, D11S917 and D11S1893 are the most relevant asthey map near the genes of interest on chromosome 11. The results, whichdemonstrate a loss in copy number of each of these microsatellitesconfirm the findings of our FISH experiments. A number of the tumor celllines have near-triploid or near-tetraploid karyotypes, thus DNA copynumber of 1 or 2 on the polyploid background reflects a loss of copynumber.

LOH analysis was performed as an independent test to validate our FISHand QuMA results. Results were available for nine of the eleven OSCCcell lines studied by FISH and QuMA. The results of LOH analysis,summarized in FIG. 2, substantiate the loss patterns we observed fordistal 11q using FISH and QuMA, as extended segments of 11q were shownto have complete or partial LOH in the “distal 11q loss without 11q13amplification” and “11q13 amplified with distal loss” OSCC cell linegroupings. The cell lines with “no 11q13 amplification, no distal loss”do not demonstrate LOH along 11q.

Distal 11q Loss Results in Chances in Expression of MRE11A, ATM, H2AXand CHEK1.

Taqman quantitative reverse transcriptase PCR (qRT-PCR) andimmunoblotting were performed to assess whether loss of one or morecopies of the MRE11A, ATM, H2AFX and CHEK1 genes translates into areduction in their expression (FIG. 3). UP3_(—)344, 348 and 700 were theNHOK controls used for the study. Overall, we observed that cell lineswith distal 11q loss generally exhibit a reduction on ATM and H2AFXexpression relative to control NHOK cell line and cell lines with nodistal 11q loss.

Protein expression in OSCC cell lines and various controls was assessedby immunoblotting (FIG. 4). Cell lines with distal 11q loss tend to havean overall lower expression level of the MRE11A, ATM and H2AX proteinscompared to those without distal 11q loss. Further, the relative trendsin protein expression correlated with the quantitative RT-PCR analysis.Thus, a genetic loss of distal 11q leads to reduction on the RNA andprotein expression for ATM, MRE11A and H2AX in a subset of OSCC.

Distal 11q Loss is Associated with Aberrant γ-H2AX Focus Formation.

Using phosphorylation of H2AX as a surrogate marker for a proficient DNAdamage response, Dr. Jason White evaluated the competency of OSCC celllines to detect double strand breaks and initiate repair by formation ofγ-H2AX foci after treatment with 2.5 Gy IR. Constitutive phosphorylationpatterns were observed in some of the cell lines (UPCI:SCC099 and 116)in the absence of any apparent overt or exogenous genetic insult. Hence,deficiencies in focus formation were assayed by several measurements,including the mean number of foci per cell, the distribution of foci in200 cells, and the percent of focus-positive cells. Fewer focus-positivecells, coupled with a reduced mean number of foci per cell relative tocontrol, are indicative of a deficient DNA damage response. Thedistribution of foci also shows a deficient response when thedistribution is skewed toward fewer signals as shown in FIG. 5A-B. Thehighest mean numbers of foci were seen in the cells with “no 11q13amplification, no distal loss” (min=5.40; max=9.45), and these weresimilar to those of the control fibroblast cell line. The cell linesthat are “11q13 amplified with distal loss” showed the lowest meanlevels of H2AX foci. When treated with IR, the mean number of foci wasmore than two-fold lower than cell lines without 11q alteration(min=3.02; max=4.64). Cell lines with “distal 11q loss without 11q13amplification” showed low to intermediate levels of γ-H2AX focusformation after IR (min=3.09; max=6.83). While there was focus formationin the untreated AT cell line (GM09607), there was essentially nodifference between the untreated and treated cells one hour followingtreatment, showing that the cells do not respond normally to IR.

Distal 11q Loss is Associated with Chromosomal Instability.

The total number of weighted aberrations per chromosome were determinedfor UPCI:SCC084, 104 and 116. Examples of metaphase spreads from each ofthese cell lines are shown in FIG. 9. Breakage was assessed 48 hpost-IR, and breaks were evident in the cell lines with loss of distal11q, irrespective of 11q13 status. Complete results are summarized inTable 4. We determined the 95% confidence intervals for each of the celllines evaluated for IR-induced breakage. There was no significantdifference in the total number of weighted aberrations per cell betweenthe control (2.52±0.97%) and treated (2.94±0.62%) populations ofUPCI:SCC116, which represents the “no 11q13 amplification, no distalloss” group. There is substantial agreement in the results forUPCI:SCC084 (C=1.43±0.51%; IR=6.68±1.40%; p<0.001) and UPCI:SCC104(C=3.15±0.76%; IR=6.68±1.05%, p<0.001), both of which have distal 11qloss. An insignificant increase in chromosomal breaks in UPCI:SCC116 inresponse to ionizing radiation could be caused due to a highly efficientDNA damage repair pathway or may be as a result of cells with increasedbreaks undergoing apoptosis. Thus, UPCI:SCC084 (11q13 amplified withdistal loss) and UPCI:SCC104 (distal 11q loss without 11q13amplification) demonstrated elevated levels of chromosomal aberrationsin response to ionizing radiation when compared to UPCI:SCC116 (no 11q13amplification, no distal loss).

TABLE 4 Summary of chromosomal aberrations in OSCC in response to IR.

Shaded areas indicate statistically significant results with ‘p’ value<0.001.

The frequency of anaphase bridges in five hundred cells was determinedand used an indicator of baseline chromosomal instability in OSCC. Thefrequencies of anaphase bridges in each cell line are summarized aspercentages in Table 4. Overall, the lowest frequency was seen in normalmale lymphocytes (0.7%). The lowest frequency among all OSCC cell lineswas seen in UPCI:SCC116 (0.96%), and the highest frequency was seen inUPCI:SCC142 (5.63%). In cell lines with no 11q13 amplification and nodistal loss, the maximum frequency (1.31%; UPCI:SCC099) was lower thanany of the frequencies observed in the “11q13 amplified with distalloss” and “distal 11q loss without 11q13 amplification” groups. In acomparison of proportions, most cell lines with distal 11q loss,irrespective of 11q13 amplification, have significantly elevated levelsof anaphase bridges (p<0.05). The comparison was made relative to normalmale lymphocytes. For two of the cell lines, UPCI:SCC078 and 122, thep-value is less than 0.1. Since all anaphase bridge frequencies weremeasured in untreated cell populations, the intrinsic level ofchromosomal instability appears to be higher in cell lines with 11qloss.

TABLE 5 Baseline anaphase bridge formation in OSCC.

Anaphase bridge formation was determined in eleven OSCC by Dr. JasonWhite. All OSCC cell lines with significantly high (p value <0.05)anaphase bridge formation at rest are highlighted in grey.

Distal 11q Loss is Associated with Radioresistance.

Clonogenic survival assays were used to detect sensitivity of OSCC tosurvival after DNA damage induced by ionizing radiation (FIG. 6 andTable 6). Results for the assay in triplicate, were grouped as “no 11q13amplification, no distal loss” (UPCI:SCC099, 116 and 182), “11q13amplified with distal loss” (UPCI:SCC078, 084, 131 and 136), and “distal11q loss without 11q13 amplification” (UPCI:SCC104, 122 and 142). Alsoincluded were normal human oral keratinocyte (NHOK) cells as normalcontrols and UPCI:SCC125 (separated due to its heterogeneity in copynumber by FISH). In the grouped analysis, nearly 60% of NHOKs survived asingle unfractionated dose of 1 Gy, and none survived a dose of 10 Gy.Similarly, the OSCC cells with “no 11q13 amplification, no distal loss”showed 56% survival at 1 Gy, and no survival at 10 Gy IR. Conversely,the OSCC cell lines grouped as “11q13 amplified with distal loss” hadnearly 83% survival at 1 Gy, and 9% survival at 10 Gy IR. The “distal11q loss without 11q13 amplification” cells had 80% survival at 1 Gy and7% survival after exposure to 10 Gy IR. The UPCI:SCC125 cells had a highsurviving fraction after 1 Gy (83.3±5.0%), and a small, but visiblesurviving fraction at 10 Gy (0.7±0.6%). Thus, cell lines with 11q13amplification and distal 11q loss (UPCI:SCC078, 084, 131 and 136) andcell lines with distal 11q loss without 11q13 amplification(UPCI:SCC104, 122 and 142) demonstrate similar resistance to ionizingradiation. In contrast, normal human oral keratinocytes (NHOK) and celllines without distal 11q loss are sensitive to ionizing radiation.

TABLE 6 Results of clonogenic cell survival in OSCC in response toionizing radiation. 11q13 Distal Mock Cell line Amp 11q loss treated 1Gy 2.5 Gy 5 Gy 10 Gy NHOK − − 100 ± 0.0 59.7 ± 2.1 30.3 ± 0.6 6.7 ± 0.60.0 ± 0.0 SCC099 − − 100 ± 0.0 58.0 ± 1.0 29.7 ± 1.5 5.7 ± 1.2 0.0 ± 0.0SCC116 − − 100 ± 0.0 55.0 ± 4.4 23.0 ± 2.6 6.0 ± 1.0 0.0 ± 0.0 SCC182 −− 100 ± 0.0 56.0 ± 5.2 32.7 ± 4.0 9.3 ± 2.1 0.0 ± 0.0 SCC104 − + 100 ±0.0 82.3 ± 4.7 55.3 ± 2.1 33.7 ± 1.2 7.7 ± 0.6 SCC122 − + 100 ± 0.0 77.0± 1.0 54.7 ± 0.6 24.0 ± 1.7 4.0 ± 1.0 SCC142 − + 100 ± 0.0 81.0 ± 4.051.7 ± 4.6 35.3 ± 1.5 9.7 ± 0.6 SCC078 + + 100 ± 0.0 83.0 ± 2.6 56.7 ±0.6 38.0 ± 1.7 7.0 ± 0.0 SCC084 + + 100 ± 0.0 80.7 ± 4.6 52.3 ± 5.0 24.7± 2.1 7.7 ± 0.6 SCC131 + + 100 ± 0.0 86.7 ± 2.5 51.7 ± 3.8 37.3 ± 2.111.3 ± 2.5  SCC136 + + 100 ± 0.0 81.7 ± 3.5 55.0 ± 4.6 34.7 ± 2.5 11.0 ±1.0  SCC125 − + 100 ± 0.0 83.3 ± 5.0 40.3 ± 3.2 27.7 ± 0.6 0.7 ± 0.6

FIG. 7A-D presents a summary of the results for distal 11q loss and itseffects in representative OSCC.

Loss of the G1 Checkpoint in a Subset of OSCC.

OSCC were treated with ionizing radiation and performed flow cytometryin order to study the cell cycle profiles of OSCC in response to DNAdamaging agents. The cell cycle profiles of different cell lines to IRare summarized in Table 7. FIG. 8 demonstrates the cell cycle profilesof two cell lines, UPCI:SCC066 and 104 in response to 5 Gy IR. Weobserved that even in untreated cells, UPCI:SCC104 has a considerablyhigh percentage of cells in the S and G2M phases. Following IR, SCC066shows accumulation of cells in both the G1 and G2M phases while,UPCI:SCC104 shows a predominant accumulation of cells in the S and G2Mphases. Thus, UPCI:SCC104 demonstrates a loss of G1 phase cell cyclecheckpoint in response to IR.

TABLE 7 Results of cell cycle analysis in OSCC in response to ionizingradiation. Untreated 5 Gy IR (24 h) UPCI: Cell Line Sub G₀ G₁ S G₂M SubG₀ G₁ S G₂M NHOK 0.1%   71% 13% 15.9%   3% 75%  2%   20% GM09607   2%52.2% 6.8%    39%   6% 34% 8.9%  51.1% SCC066 1.2%   70% 13.3%   15.5%1.9% 50.3%   16.1%   31.7% SCC084 0.7%   60% 10% 29..3%  2.4% 32% 12%53.6% SCC104 0.9%   53% 16% 30.1% 2.8% 22% 18% 57.2% SCC105 0.7%   67%13% 19.3%   4% 51% 15%   30% SCC116 1.1% 61.9% 15%   22%   4% 49% 15%  32% SCC131   1% 66.5% 15.5%     15% 2.5% 32% 11% 55.5% SCC136   1%59.9% 10% 29.1% 1.9% 31.6 25.5   41% SCC142 1.3% 62.2% 14.2%   22.3%2.6% 31% 16% 40.4%Thus a loss of G1 checkpoint was observed in response to IR in 5 out of8 OSCC studied. Even UPCI:SCC066, 105 and 116 with an intact G1checkpoint, had an increased accumulation of cells in the G2M phasecompared to NHOK. This may be either due to higher number of cells inthe G2M checkpoint at the time of DNA damage or due to cell line/tumorheterogeneity wherein a fraction of cells in UPCI:SCC066, 105 and 116lack p53 and are deficient in their G1 checkpoint. A loss of G1checkpoint in OSCC leads to increased number of cells with unrepairedDNA damage entering the S and the G2M phases of the cell cycle. Thus,OSCC with an enhanced S and G2M phase cell cycle checkpoints are able toavoid p53-independent cell death (PCC/MC) and have a growth advantage.

p53 and ATR Expression.

It was observed that loss of p53 protein expression is associated withoverexpression of ATR in a larger panel of OSCC cell lines (FIG. 9).Thus, it is possible that either loss of p53 leads to ATR upregulation,or overexpressed ATR in OSCC may affect p53 regulation and function.Carcinomas with loss of the G1 checkpoint express loss of p53 proteinexpression and ATR/CHEK1 overexpression at the RNA and protein levels

p53 activity and/or expression varies among cancer cell lines. FIGS. 32Aand B show respectively, the expression of these proteins in oralsquamous carcinoma and non-small cell lung carcinoma and ovariancarcinoma cell lines, respectively. Active p53 results in induction ofp21 expression. The extent of radiosensitivity of cancer cellsexpressing active p53 (for example, A549 (FIG. 32B), which showsinduction of p21) was observed to be somewhat greater than that of cellsthat did not contain active p53 (for example, ES-2 (FIG. 32B) andUPCI:SCC040); compare the results shown in FIG. 34 (active p53) withFIGS. 33 and 35 (no active p53). As shown in FIG. 35, cells lackingactive p53, although somewhat less sensitive to radiation, respondeddramatically to a combination of irradiation and inhibition of CHEK1 byCHEK1 siRNA, cell survival being drastically reduced

Copy Number and Structural Changes Involving the ATR and CHEK1 Genes.

To determine if there are structural or numerical changes in the ATR andCHEK1 genes in OSCC, dual-color FISH with BAC probes to ATR wasperformed along with a centromere 3 enumeration probe (CEP 3) and BACprobes to CHEK1 and compared it to centromere 11 enumeration probe (CEP11; D11Z1) (Vysis, Downers Grove, Ill.). Table 8 summarizes the FISHresults in terms of copy number gain in relation to the ploidy of eachcell line, which was derived from chromosome 3 centromere enumerationand consensus karyotypes. Copy number gains were observed in the ATRgene in all cell lines with distal 11q loss. In addition, it wasobserved that in a number of cell lines depicted in FIG. 10A-F and Table8 that copy number gains were associated with isochromosome 3qformation. In UPCI:SCC084, copy number gain in the ATR gene was notobserved. However, by metaphase FISH, a translocation of one copy of theATR gene was detected. On further evaluation of UPCI:SCC084, the ATRgene was translocated to a derivative chromosome 11 with 11q13amplification. In addition to OSCC, low level amplification (8-10 copiesper cell) was observed in an ovarian tumor cell line, Ovcar-3. It wasobserved that CHEK1 was partially lost in all OSCC cell lines with 11q13amplification. Loss of CHEK1 was also seen in UPCI:SCC122 and 142 whichdid not demonstrate 11q13 amplification. Of the twenty OSCC cell linesstudied, ATR gain was observed in ten OSCC and CHEK1 loss in fifteenOSCC suggesting that both these events occur frequently in OSCC.

TABLE 8 Results of FISH analysis for the ATR and CHEK1 genes. ATR CHEK1% cells % cells 11q13 ATM % cells % cells with no ATR:CEP3 Iso % cells %cells with no CHEK1:CEP11 Cell line Amp loss with gain with loss changeratio 3q with gain with loss change ratio Normal — — 100%  1.0 − — —100% 1.0 SCC003 + + 53% 1% 46% 1.6 − 1% 93% 6% 0.71 SCC029B + + 89% 2% 9% 1.4 − — 99% 1% 0.61 SCC032 + + 57% — 43% 1.3 − 1% 99% — 0.67SCC040 + + 53% — 47% 1.6 − 5% 94% 1% 0.56 SCC066 − −  2% 3% 95% 1.0 − 1% 1% 98% 1.01 SCC070 + + 89% 1% 10% 2.1 + — 98% 2% 0.57 SCC077 + + 85% 1%14% 2.2 + 2% 31% 67% 0.67 SCC078 + +  9% 3% 88% 1.0 − — 94% 6% 1.05SCC084 + +  7% — 93% 1.0 − 2% 98% — 0.52 SCC099 − −  3% 6% 91% 1.0 − —100%  — 1.0 SCC103 + + 91% 1%  8% 1.1 − 2% 94% 4% 0.74 SCC104 + + 98% — 2% 2.2 + 1% 11% 88% 0.97 SCC105 − + 2.5%  0.5%   97% 1.1 − 12%  32% 56%0.85 SCC116 − −  3% 2% 95% 1.0 − 3% 10% 87% 0.94 SCC122 − +  1% 9% 90%1.0 − 1% 82% 17% 0.71 SCC131 + + 92.5%   — 7.5%  2.1 + — 98% 2% 0.44SCC136 + + 78.5%   — 21.5%   1.3 − — 96% 4% 0.53 SCC142 − + 98.5%   —1.5%  2.1 + — 91% 9% 0.67 SCC172 + +  3% 5% 92% 1.1 − 5% 90% 5% 0.71SCC182 − −  1% 1% 98% 1.0 − 3% — 97% 1.01 MDA231  6% 4% 90% 1.0 − 5%  1%94% 1.1 MCF7 — 1% 99% 1.0 − —  3% 97% 1.0 SK-OV3  4% 3% 93% 1.1 − 1% —99% 1.0 OVCAR3 99% —  1% 9 − 8% — 92% 1.1Five primary squamous cell carcinomas of the oral cavity were tested forATR and CHEK1 copy number alterations (Table 9, FIG. 11A-F). A gain incopy number for the ATR gene was detected in the tumor tissue in allfive tumors while the surrounding normal tissue exhibited normal copynumber for the ATR gene and CEP 3. Similarly, CHEK1 was partially lostin 35% to 65% of cells in the tumor tissue, while the adjacent normaltissue showed no loss of the gene. High level CCND1 amplification wasfound in 65% to 100% of the tumor cells suggesting that 11q13amplification may be an early change. Thus, ATR gain and CHEK1 loss arepresent not only in OSCC cell lines but also in the head and neckprimary tumors.

TABLE 9 ATR, CHEK1 and CCND1 FISH in primary head and neck tumors. ‘T’represents the head and neck primary tumor and ‘N’ representssurrounding normal tissue. Figures in each column represent % cells withgain, % cells with loss or % cells with no change. ATR CHEK1 CCND1 Amp/Amp/ Amp/ Sample I D: Gain Normal Loss Gain Normal Loss Gain Normal Loss0402584 N 2% 98% — — 99% 1% 2% 98% — T 54% 46% — — 35% 65% 100% — —3L621340 N 1% 99% — — 98% 2% 1% 99% — T 58% 42% — — 65% 35% 99% 1% —1H620781 N 2% 98% — — 98% 2% 3% 94% 3% T 32% 66% 2% — 51% 49% 65% 34% 1%3G040281 N 1% 98% 1% 1% 98% 1% — 100% — T 42% 58% — — 44% 56% 100% — —1F620600 N — 98% 2% — 99% 1% 3% 95% 2% T 42% 57% 1% — 37% 63% 68% 32% —

Mechanism of ATR Gain and ATR Translocations.

It has been previously reported that in OSCC, translocations ofchromosome 3 are commonly associated with 11q13 amplification andfrequently, chromosome 3 fragments cap the amplified chromosome 11. Itwas observed that three of 20 OSCC cell lines studied (UPCI:SCC078, 084and 172) had a translocation between the derivative chromosome 11der(11) with 11q13 amplification and segments of 3q along with the ATRgene (FIG. 12A, B). Similar translocations between chromosome 3 andder(11) with 11q13 amplification in OSCC were also reported by anothergroup, suggesting that this event may be common in head and neck tumors(JIN et al. 2002). In all three OSCC cell lines, UPCI:SCC078, 084 and172, this t(3;11) translocation was present in all twenty metaphasesstudied.

Isochromosome 3q formation was found in five of 20 OSCC cell lines(UPCI: SCC070, 077, 104, 131 and 142) studied. Isochromosome 3qformation is a common mechanism of 3q and ATR gain in OSCC.Isochromosome formation can occur either through centromere splitting orby fusion of two chromosome 3 with breaks at 3p resulting in aniso-dicentric chromosome 3. Isodicentric chromosomes 3q was observed inSCC070, 077, 104, 131 and 142, suggesting that centromere splitting isnot the mechanism by which isochromosome formation occurs in OSCC (FIG.12, C).

Dicentric chromosomes are prone to being pulled to opposite centromeresduring cell division resulting in an anaphase bridge between the twodaughter cells. The formation of a dicentric chromosome 11 is also anintermediate in the process of 11q13 amplification and its presence inanaphase bridges may indicate a stage of ongoing amplification andchromosomal instability. The frequency of ATR gene and CEP 3 presencewas observed in anaphase bridges and compared it to the presence of CEP11 in anaphase bridges in a subset of four OSCC cell lines and GM09607(AT cell line). The results for 50 anaphase bridges analyzed for eachcell line are summarized in Table 10 (see also FIG. 13A-B). An increasedfrequency of ATR gene was observed in anaphase bridges in UPCI:SCC104and 131, both of which have gain for chromosome 3, but not inUPCI:SCC066 and 105 which have normal copy numbers for the ATR gene.Surprisingly, an increase in the frequency of ATR gene present wasdetected in anaphase bridges in GM09607, which does not have 3q and ATRgain. Since GM09607 relies heavily on the upregulated ATR-CHEK1 pathwayfor survival, anaphase bridges may serve as a mechanism for formation ofisodicenteric 3q leading to ATR and 3q gain. Thus, the high frequency ofATR and CEP3 in anaphase bridges may represent ongoing selection for 3qgain. On the other hand, chromosome 3, on account of its large size maybe prone to be present in anaphase bridges at an increased frequency.

TABLE 10 Frequency of ATR, CEP 3 and CEP11 in anaphase bridges in OSCCand GM09607. 11q13 Frequency of Frequency of Frequency of amplifi- ATRATR in CEP3 in CEP11 in Cell Line cation gain bridges bridges bridgesSCC066 − −  4%  4%  6% SCC104 − + 26% 26% 14% SCC105 − −  6%  6% 22%SCC131 + + 28% 28% 24% GM09607 − − 16% 16% 10%

ATR and CHEK1 are Overexpressed in a Subset of OSCC.

To evaluate if changes in copy number for ATR and CHEK1 genes in OSCClead to changes in expression, ATR and CHEK1 RNA expression was studiedusing qRT-PCR and protein expression by immunoblotting. Our qRT-PCRresults are summarized in FIG. 14A-C. Since the RNA expression wasmeasured relative to universal reference with diploid chromosomalconstitution, a relative expression <1.5 in OSCC (with near-triploid totetraploid karyotypes) can be considered as a reduction in relative RNAexpression.

RNA expression was evaluated by qRT-PCR in eleven OSCC cell lines,GM09607 (AT cell line). NHOK and HEK 293 cells were used as control celllines. UPCI:SCC066, 099, 105 and 122 showed ATR and CHEK1 expressionequal to or lower than the NHOK and HEK293 control cell lines. GM09607which has been shown to have an upregulated ATR-CHEK1 pathwaydemonstrated 4-fold increase in ATR expression and 8-fold increase inCHEK1 expression. Six out of eleven cell lines including UPCI:SCC040,104, 131, 136, 142 and 172 demonstrated an increase in both ATR andCHEK1 expression. Around 8-fold increase in ATR expression and 18-20fold increase in CHEK1 expression were observed in two OSCC cell lines:UPCI:SCC040 and 104. It should be noted that all cell lines with ATMloss (UPCI:SCC 104, 131, 136 and 142) other than UPCI:SCC122demonstrated a significant increase in ATR and CHEK1 RNA expression.

Immunoblotting for ATR and CHEK1 proteins shown in FIG. 15 confirmed theqRT-PCR results. An increase in the expression for ATR was found in allthe cell lines with copy number gain for the ATR gene. ThoughUPCI:SCC084, the cell line with the highest basal expression of ATR didnot have any gain of the ATR gene, it had a translocation of one of itscopies of the ATR gene which may account for the increased expression.

Earlier reports suggested that kinase-dead ATR can inhibit serine15-p53phosphorylation in response to DNA damage and thus, block p53 activationin response to IR and UV radiation (TIBBETTS et al. 1999). To checkwhether the overexpressed ATR in UPCI:SCC084 and 104 was kinase-dead,CHEK1 phosphorylation on serine-345 was assayed following treatment withIR and ultra-violet radiation (UV). Since CHEK1 s345 phosphorylationfollowing UV radiation is preferentially mediated through ATR kinase, itappears that ATR kinase activity is not lost in any of the four celllines studied (FIG. 16A-C). On studying ATR mediated phosphorylation ofSMC1 on serine 957, in response to 20 J/m2 UV radiation, UPCI:SCC084 and104 were found to have much higher levels of SMC1 phosphorylation. SinceSMC 1 phosphorylation occurs during the S and G2M phases, these resultsconfirm findings that the G1 is compromised in SCC084 and 104. Increasedphosphorylation of Cdc25C on serine-216 was observed in response to 5 GyIR in UPCI:SCC104 compared to SCC066. These observations suggest that anoveractive ATR-CHEK1 response is present in UPCI:SCC084 and 104 and thata higher number of cells with DNA damage enter the S and G2M phases inresponse to DNA damage.

An Upregulated ATR-CHEK1 Pathway is Associated with Radioresistance.

It was observed that all cell lines with an upregulated ATR-CHEK1pathway demonstrate increased resistance to ionizing resistance (Table11). Since loss of p53 was also observed in these cell lines, theresistance to ionizing radiation could be as a result of loss ofp53-mediated apoptotic pathways and an upregulated ATR-CHEK1 pathwaywhich promotes G2M accumulation and HRR after DNA damage.

TABLE 11 Clonogenic cell survival of OSCC to different doses of IR ATR,CHEK1 Mock Cell line overexpression treated 1Gy 2.5Gy 5Gy 10Gy NHOK −100 ± 0.0 59.7 ± 2.1 30.3 ± 0.6  6.7 ± 0.6 0.0 ± 0.0 SCC084 + 100 ± 0.080.7 ± 4.6 52.3 ± 5.0 24.7 ± 2.1 7.7 ± 0.6 SCC104 + 100 ± 0.0 82.3 ± 4.755.3 ± 2.1 33.7 ± 1.2 7.7 ± 0.6 SCC142 + 100 ± 0.0 81.0 ± 4.0 51.7 ± 4.635.3 ± 1.5 9.7 ± 0.6 SCC131 + 100 ± 0.0 86.7 ± 2.5 51.7 ± 3.8 37.3 ± 2.111.3 ± 2.5  SCC136 + 100 ± 0.0 81.7 ± 3.5 55.0 ± 4.6 34.7 ± 2.5 11.0 ±1.0  SCC066 − 100 ± 0.0 56.7 ± 1.1 29.5 ± 2.2  5.5 ± 1.0 0.0 ± 0.0SCC105 − 100 ± 0.0 60.1 ± 2.1 32.3 ± 1.5  7.9 ± 2.6 0.0 ± 0.0

Caffeine a Non-Specific Kinase Inhibitor Sensitizes OSCC to IR InducedDNA Damage.

In order to study the relative contribution of ATR and CHEK1overexpression to the radioresistance observed in a subset of OSCC, wedetermined the sensitivity of OSCC to caffeine which is a non-specificinhibitor of ATM and ATR. OSCC were treated with caffeine in combinationwith DNA damaging agents (IR or aphidicolin). The following tests wereperformed to estimate the sensitivity of OSCC to caffeine: the sub-G0population in flow cytometeric studies, the surviving fraction usingclonogenic cell survival assay and PCC/MC formation in response tocaffeine.

A prolonged S phase and G2M accumulation were observed in a subset ofirradiated OSCC cells which suggests that the S and G2M checkpoint areenhanced in IR treated OSCC. One possible explanation for thisobservation would be that the G2M accumulation in irradiated OSCC wasmediated through the ATR-CHEK1 pathway, as seen in irradiated AT cells(WANG et al. 2004). OSCC was treated with caffeine, a non-specificinhibitor of ATM and ATR kinases and evaluated their cell cycle profilesfollowing irradiation (FIG. 17). It was found that caffeine clearlyreduced accumulation of cells in the S and G2M phases and increased thesub-G0 population (dead cells) in cells lines with an upregulatedATR-CHEK1 pathway including GM09607 (AT cells). In comparison, celllines with an intact G1 checkpoint and normal ATR-CHEK1 expression didnot demonstrate any sensitivity to caffeine.

To detect the mechanism of caffeine-induced cell death, ‘S’ phase PCCand MC formation was evaluated in response to only aphidicolin (0.4 μM),or in cells pretreated with 1 mM caffeine 1 h before aphidicolintreatment (FIG. 18A-C). Criteria for distinguishing PCC/MC from normalmetaphase were adapted from a previous report (NGHEIM et al. 2001). Avery high percentage of mitotic cells with PCC/MC in UPCI:SCC084 and 104were observed if pretreated with 1 mM caffeine (FIG. 18A). This highpercentage was comparable with GM0607 (AT cells), which was used as apositive control. In comparison, UPCI:SCC066 and 105 with an intact G1checkpoint demonstrate low levels of PCC/MC formation even aftercaffeine pretreatment. The morphology of PCC/MC is depicted in FIG. 18B.Thus, caffeine mediated elimination of the G2M peak is caused due toincreased death (by PCC/MC) of these cells by entering premature S phaseor premature mitosis.

It was found that cell lines with upregulated ATR-CHEK1 pathway hadprolonged G2M accumulation following irradiation and a reducedsensitivity to IR-induced cell death. However, the exact mechanism ofhow an enhanced G2M checkpoint can promote radioresistance is not known.It is likely that prolonged G2M accumulation may allow adequate time forrepair and thus promote cell survival. Since caffeine eliminates the G2Maccumulation observed in OSCC, the sensitivity of OSCC to differentdoses of caffeine was studied by clonogenic cell survival. It wasobserved that caffeine enhances cell sensitivity to ionizing radiation,especially in OSCC with upregulated ATR-CHEK1 pathway (FIGS. 19, 20).UPCI:SCC084 and 104 showed a significant (>50%) reduction in colonysurvival at a dose of 0.5 mM caffeine and complete absence of survivalat 1 mM caffeine. Thus, OSCC with an upregulated ATR-CHEK1 pathway(UPCI:SCC084 and 104) exhibit increased sensitivity to caffeine in theabsence of any DNA damaging agents.

ATR and CHEK1 siRNA Sensitize a Subset of OSCC to Ionizing Radiation andAphidicolin Induced DNA Damage.

Small inhibitory RNA (siRNA) are composed of 21-25 nucleotides which arecomplimentary to a known ‘target’ mRNA (ELBASHIR et al. 2001). Usually apool of two or more siRNA duplexes is used to specifically bind to anddegrade the ‘target’ mRNA. Usually peak siRNA transfection is seen atthe end of 48-72 hrs and the mRNA knockout lasts for 1-3 weeks dependingon the type and stability of the siRNAs. Recently, there is growinginterest to use siRNA mediated gene targeting to inhibit specific genesinvolved in cancer and other diseases (WALL and SHI 2003).

In the following experiments, siRNA specific to ATR and CHEK1 was usedto reduce their expression in UPCI:SCC066 and 104. Transfectionefficiency was calculated using a non specific siRNA labeled with afluorescent tagged siGLO (Dharmacon). Reduction in ATR and CHEK1expression was determined by checking protein levels by immunoblottingat the end of 72 h. The results of these experiments were a high levelof ATR and CHEK1 knockout by their specific siRNAs while a scramblednon-specific siRNA did not inhibit ATR and CHEK1 protein expression(FIG. 21).

The cell cycle profiles of UPCI:SCC066 and 104 were analyzed after ATRand CHEK1 knockout using the respective siRNAs (FIG. 22A-B). Even in theuntreated sample, there were a very high number of cells in the S (18%)and G2M (29%) phases in SCC104. Following treatment with 5 Gy IR, at theend of 24 h, a very high percentage of cells (nearly 55%) accumulated inthe G2M phase in SCC104 compared to 31% in SCC066. Following treatmentwith ATR siRNA, in irradiated SCC104, there was a significant reductionin the G2M accumulation of cells from 55% to 18% and an increase in thesub-G0 population (dead cells) from 1% to nearly 18%. Since acorresponding increase occurred in the sub-G0 population, the reductionof the G2M peak observed in SCC104 appears to be due to cell death andnot due to a reduction in the number of cells that enter G2M. Even inun-irradiated SCC104 cells, there was a reduction in the G2Maccumulation and an increased sub-G0 population (15%). In comparison,un-irradiated as well as irradiated SCC066 cells demonstrated a modestreduction in G2M accumulation following treatment with ATR siRNA but nocorresponding increase in the dead (sub-G0) cell population. A similarsensitization of UPCI:SCC104 but not UPCI:SCC066 was observed followingtreatment with CHEK1 siRNA. Following treatment with a non-specificscrambled siRNA, SCC104 does not exhibit any reduction in the G2Maccumulation or increase in the sub-G0 population. This suggests thatthe effects observed were specific for ATR and CHEK1 inhibition.

Thus, UPCI:SCC104 was highly sensitized to IR following inhibition ofthe upregulated ATR-CHEK1 pathway with the respective siRNAs. Theseresults suggest that SCC104 with ATR and CHEK1 overexpression is highlysusceptible to IR, following treatment with ATR or CHEK1 siRNA.

To determine if the PCC/MC formation associated with caffeine treatmentwas mediated through its inhibition of the ATR-CHEK1 pathway, ATR andCHEK1 siRNAs were used to produce a specific knockout. A very high levelof PCC/MC in UPCI:SCC104 was detected following treatment with eitherATR or CHEK1 siRNA (FIG. 23). In comparison UPCI:SCC066 had a modestincrease in the number of cells undergoing PCC/MC in response toinhibition of the ATR-CHEK1 pathway.

Finally, cell survival of UPCI:SCC066 and 104 after treatment with ATRsiRNA was studied in the absence of any DNA damaging agents (FIG.24A-B). Complete inhibition of colony formation was observed in SCC104treated with ATR siRNA at the end of 12 days. In comparison, UPCI:SCC066did not demonstrate a significant reduction in cell survival.

Thus, these results suggest that the ATR-CHEK1 pathway is upregulated ina subset of OSCC and ATR and/or CHEK1 may be potential targets tosensitize a subset of OSCC to DNA damaging agents.

7. EXAMPLE Loss of Distal 11q is Associated with DNA Repair Deficiencyand Reduced Sensitivity to Ionizing Radiation

7.1 Materials and Methods

Cell Culture.

HNSCC cell lines, normal human oral keratinocytes (NHOK), and normalhuman fibroblasts were established in the inventors' laboratory.hTERT-transfected human oral keratinocytes were OKF6/TERT-1 from thelaboratory of Dr. James G. Rheinwald; Dickson et al., 2000. GM09607, anATM null cell line, was from Coriell Cell Repositories, Camden, N.J. TheHNSCC cell lines were grown in Minimal Essential Medium (GibcoInvitrogen, Grand Island, N.Y.), supplemented with 1% non-essentialamino acids, 1% L-glutamine, 0.05 mg/ml Gentamicin and 10% FBS) (allsupplements from Gibco Invitrogen) (White et al., 2006). NHOKestablished from uvulopalatopharyngoplasty specimens were cultured inserum-free KGM-2 medium (Clonetics, Walkersville, Md.), supplementedwith BPE (bovine pituitary extract), hEGF, insulin (bovine),hydrocortisone, GA-1000 (Gentamicin, Amphotericin B), epinephrine andtransferrin as per the manufacturer's instructions (supplements suppliedin the KGM-2 BulletKit™ from Clonetics). Finite passage normal humanfibroblasts established from uvulopalatopharyngoplasty tissue werecultured in Minimal Essential Medium (Gibco Invitrogen), supplementedwith 1% non-essential amino acids, 1% L-glutamine, 0.05 mg/ml Gentamicinand 10% FBS) (Gibco Invitrogen). The OKF6/TERT-1 cells were initiallycultured in Keratinocyte-SFM supplemented with 25 μg/ml bovine pituitaryextract, 0.2 ng/ml epidermal growth factor, 0.3 mM CaCl2 andpenicillin-streptomycin (Gibco Invitrogen). These keratinocytes wereexpanded to high density in a 1:1 mixture of Keratinocyte-SFM andDMEM-F12. The DMEM-F12 was a 1:1 mixture of calcium-free andglutamine-free DMEM and Ham's F-12 supplemented with 25 μg/ml bovinepituitary extract, 0.2 ng/ml epidermal growth factor, 1.5 mM L-glutamineand penicillin-streptomycin (Gibco Invitrogen). GM09607 (Coriell CellRepositories, Camden, N.J.) was cultured in Dulbecco's Modified EagleMedium (DMEM), supplemented with 1% non-essential amino acids, 0.05mg/ml penicillin-streptomycin-L-glutamine, and 10% FBS (GibcoInvitrogen).

Fluorescence In Situ Hybridization (FISH).

To prepare mitotic cells for FISH analyses, HNSCC cells were harvestedfollowing a 5 h treatment with 0.1 μg/ml Colcemid™ (Irvine Scientific,Santa Ana, Calif.), hypotonic KCl (0.075M) treatment for 16 min, andfixation in 3:1 methanol:glacial acetic acid. All other cells wereharvested using the same method, except that 1 h of Colcemid™ waspreferred for non-tumor cells. FISH analysis was used to detect copynumber changes in the CCND1, MRE11A, ATM and H2AFX genes in the HNSCCcell lines. For FISH analysis, cells were harvested, dropped ontoslides, treated with RNase/2×SSC, and dehydrated using a graded series(70%, 80% and 100%) of ethanol washes. Chromatin was denatured with 70%formamide and the cells were dehydrated in a second graded series ofethanol washes. The CCND1, ATM, MRE11A and H2AFX probes for FISH wereprepared following DNA extraction from BACs purchased from Children'sHospital of Oakland Research Institute (CHORI, Oakland, Calif.). The BACDNA was isolated and labeled using a nick translation kit fromVysis/Abbott Molecular Inc. (Des Plaines, Ill.). The labeled DNA wasprecipitated with ethanol, resuspended in hybridization buffer,denatured for 5 min at 75° C., and preannealed for 15-30 min at 37° C.Each probe was hybridized for 16 h at 37° C., after which slides werewashed with SSC/Tween-20. Slides were counterstained with DAPI andmounted with antifade prior to analysis. All FISH analyses were carriedout using an Olympus BX-61 epifluorescence microscope (OlympusMicroscopes, Melville, N.Y.). An Applied Imaging CytoVision workstationwith Genus v3.6 software was used for image capture and analysis(Applied Imaging, San Jose, Calif.).

Paraffin and Frozen Section FISH.

Anonymous frozen sections of breast carcinomas and paraffin sections ofthe stage III ovarian carcinomas were obtained from the Magee-WomensHospital Tissue Procurement Program. The paraffin sections of anonymousHNSCC were obtained from the Head and Neck SPORE Tissue Bank at theUniversity of Pittsburgh. 4-5 μM thick sections of formalin-fixed,paraffin-embedded tissue were mounted on positively charged microscopeslides. The slides were aged overnight at 60° C., following which theywere deparaffinized twice with xylene for 5 min each at roomtemperature. The slides were dehydrated in two 100% ethanol washes, 2min each at room temperature. The slides were then treated with0.5×SkipDewax solution (Insitus Biotechnologies, Albuquerque, N. Mex.)at 80° C. for 15 min followed by a wash in distilled water for one minat room temperature, treatment in 0.2N HCl for 20 min at roomtemperature, pretreatment with 1 M sodium thiocyanate at 80° C. for 30min, a wash in distilled water for one min at room temperature, twowashes with 2×SSC 5 min each, and then treatment with protease (pepsin)solution for 15 min at 37° C. Next, the slides were fixed in 10%Formalin for 10 min at room temperature. After two 2×SSC washes at roomtemperature, the slides were dehydrated with a graded series of ethanolwashes at room temperature, and allowed to dry on slide warmer. Next,slides were denatured at 75° C. in 70% formamide, followed by a coldseries of ethanol washes. The probes for paraffin FISH were prepared asdescribed for FISH above. The hybridization of the FISH probes andpost-hybridization treatment of the paraffin sections was carried out asdescribed for FISH above.

4-5 μM thick sections of OCT-embedded, fresh frozen breast carcinomaswere mounted on positively charged microscope slides. The slides wereplaced in 0.8% sodium citrate for 20 min, fixed in 3:1 methanol aceticacid, and air dried. The slides were aged at 90° C. for 10 min, digestedin 0.005% pepsin in 0.2N HCl at 37° C. for 60 sec, incubated in 70%ethanol for 30 sec, pretreated in 2×SSC at 37° C. for 60 min, dehydratedin a graded series of ethanol, denatured and hybridized as for FISHabove.

Quantitative Microsatellite Analysis (QuMA).

QuMA analysis to detect copy number changes in microsatellite markersalong chromosome arm 11q was carried out as described in Huang et al.,2002, 2006. PCR primer sequences for the microsatellite loci are shownin Appendix D. The TaqMan CA-repeat fluorogenic probe used for all lociconsisted of the following sequence: 5′-FAM (6-carboxyfluorescein)-TGTGTGTGTGTGTGTGTGTGT-TAMRA(6-carboxytetramethylrhodamine)-3′ (SEQ ID NO:26). All of the probes andprimers for QuMA were purchased from Integrated DNA Technologies(Coralville, Iowa).

Loss of Heterozygosity.

For the LOH studies, DNA was isolated from HNSCC cell lines by standardSDS/proteinase K/phenol-chloroform extraction methods. Genotypes of thecell lines were compared to constitutional DNA isolated from thepatient's blood. Microsatellite loci for chromosome arm 11q were chosenfrom published maps, and samples were PCR-amplified using standard 32Ptechniques. Standard cycling conditions for LOH were as follows:following denaturation at 94° C. for 5 min, samples were subjected to 35cycles of 94° C. for 45 sec, 55° C. for 45 sec and 72° C. for 45 sec.Amplified products were electrophoresed through a 6% denaturingpolyacrylamide gel with appropriate size standards, before exposure tox-ray film. Complete or nearly complete loss (≧90%) of an allele at agiven locus was called “LOH.” Allelic loss greater than 50% was called“partial LOH,” and allele loss less than 50% was not considered (“noLOH”).

Quantitative Reverse Transcriptase PCR(RT-PCR).

RT-PCR was performed to detect expression of MRE11A, ATM and H2AFX inHNSCC. Taqman primers and probes were designed with Primer ExpressV.2.0.0 (Applied Biosystems, Foster City, Calif.). The reversetranscriptions were carried out as previously in Huang et al., 2002.Quantitative PCR (QPCR) was performed on the cDNA using the ABI 7700Sequence Detection Instrument (Applied Biosystems) and analyzed usingthe relative quantitation method (User Bulletin, PE Applied Biosystems)as in Huang et al. (2002). QPCR was carried out for MRE11A, ATM, H2AFXand ribosomal 18S RNA (endogenous control) with primers and probeslisted in Appendix D. For the QPCR, the final concentrations of thereaction components were as follows: PCR buffer A (Applied Biosystems),300 nM each dNTP, 3.5 mM MgCl2, 0.06 units/μl Amplitaq Gold (AppliedBiosystems), 500 nM (100 nM for 18S RNA) primers, and 200 nM (100 nM for18S RNA) probe. The thermocycler conditions were 95° C. Taq activationfor 12 min and 40 cycles (30 cycles for 18S RNA) of 95° C. denaturationfor 15 sec followed by 60° C. anneal/extend (64° C. for MRE11A andH2AFX) for 60 sec.

Immunoblotting.

Immunoblotting was utilized to detect protein expression of MRE11A, ATMand H2AX in HNSCC cell lines, and also to assess the phosphorylationlevels of H2AX following exposure to 2.5 Gy IR. Flasks of each cell linewere trypsinized, washed with ice cold 1×PBS and lysed on ice with asolution containing 50 mM Tris, 1% Triton X-100 (Sigma), 0.1% sodiumdodecyl sulfate (Bio-Rad Laboratories, Hercules, Calif.), 150 mM NaCl(Fisher Chemicals, Fairlawn, N.J.), 1 mM dithiothreitol (DTT) (FisherScientific, Inc., Hampton, N.H.), 10 μg/ml leupeptin (Roche AppliedScience, Indianapolis, Ind.), 10 μg/ml pepstatin (Roche Applied Science)and 1 nM phenyl methyl sulfonyl fluoride (PMSF) (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.). The soluble cell lysate wascentrifuged at 2000×g for 15 min, and transferred to a clean microfugetube. Histone H2AX was extracted from the remaining pellet containinginsoluble protein and chromatin. The pellet was treated with 0.1N (3.65g/L) HCl for 20 min on ice, and then centrifuged at 10,000×g for 10 min.

Protein concentrations were determined using the Bio-Rad Quick StartBradford Protein Assay Kit and the SmartSpec 3000 (Bio-RadLaboratories). The acid lysate was neutralized with Tris-EDTA (TE) pH8.0 prior to normalization. Normalized lysates were resolved by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andtransferred onto an Immobilon-P membrane (Millipore Corporation,Billerica, Mass.). After blocking with 5% non-fat dry milk (NFDM) for 1h, the membrane was incubated overnight with antibodies for ATM (5C2,used at 1:1000), MRE11A (C-16, used at 1:1000), total H2AX (used at1:1000) or mouse anti-phospho-Histone H2AX (Ser139, clone JBW301, usedat 1:1000) at room temperature (ATM and MRE11A from Santa CruzBiotechnology, Inc.; total H2AX from Cell Signaling Technology, Inc.,Danvers, Mass.; γ-H2AX from Upstate Technology, Inc., Lake Placid,N.Y.). Following three 5 min washes in 1×TBST (Tris-buffered SalineTween-20), the membrane was incubated with the appropriate secondaryantibody (1:3000) for 2 h. Target proteins were visualized using theWestern Lightning™ Chemiluminescence Reagent Plus kit (PerkinElmer LifeSciences, Boston, Mass.) according to the manufacturer's instructions.To verify equal protein loading in the gels, membranes were stripped andre-probed with antibodies against β-actin (Sigma Immunochemicals, St.Louis, Mo.) or α-actinin (Sigma Immunochemicals) (all at a 1:1000dilution). A densitometric analysis was carried out using Un-Scan-ItGel™ (Silk Scientific, Inc., Orem, Utah).

Immunofluorescence.

Changes in γ-H2AX focus formation after exposure to 2.5 Gy IR weredetermined using Gammacell 1000 Elite irradiator (Nordion International,Inc., Ottawa, Canada) with a 137Cs source at a dose rate of 4.42 Gy/min.Each cell line was passaged into single-well chamber slides (Nalge NuncInternational, Rochester, N.Y.) one day prior to treatment with ionizingradiation. A parallel set of slides was mock-treated (medium changeonly) as a control. Following exposure to IR, the cells were washed with1×HBSS (Irvine Scientific), the medium was replaced, and the chamberslides were incubated under standard culture conditions for 1 h to allowfor repair. After repair, the cells were washed with 1×PBS, fixed with4% paraformaldehyde (PFA) (Sigma) for 30 min and permeabilized with 0.2%triton/1×PBS. Following permeabilization, cells were blocked with 5%goat serum/1×PBS and incubated with anti-phospho-H2AX primary antibody(Upstate Technology, Inc.) for 2 h. The primary antibody was detectedwith a goat anti-mouse Alexa 488 (Molecular Probes, Eugene, Oreg.) for 1h. Cells were then washed, counterstained with DAPI, mounted usingantifade and analyzed under an epifluorescence microscope as previouslydescribed. A minimum of 100 cells were scored from control andIR-treated chamber slides for each of the cell lines by two independentobservers for a total of at least 200 cells. An additional two datasets,each including a minimum of 200 cells, were accumulated using aspot-counting algorithm on a MetaSystems Metafer scanning system (trialuse courtesy of MetaSystems, Altlussheim, Germany). The mean level offocus formation was calculated using the three independent samples, and95% confidence intervals were generated to illustrate the distributionin the data.

Viability Assay.

To assay the fraction of cells undergoing apoptosis at the time oftreatment, the Reduced Biohazard Cell Viability/Toxicity Kit (MolecularProbes) was used on chamber slides (Nalge Nunc International) set up inparallel with slides made for immunofluorescence visualization of foci.In brief, slides were treated with identical doses of IR, and allowed torepair for 1 h before being washed in HEPES buffer (Irvine Scientific),and incubated in 1:500 dilutions each of SYTO 10 green fluorescentnucleic acid stain and DEAD Red ethidium homodimer-2 in HEPES buffer for15 min. Slides were mounted in 1×PBS and visualized using an Olympusepifluorescence microscope at 100× magnification. Viability wasdetermined by cellular staining, wherein membrane-competent cellsappeared green, and the cells with compromised cellular membranesappeared red. Results for the assay, carried out in triplicate, arereported as mean percentage of surviving cells with 95% confidenceintervals.

Anaphase Bridges

Anaphase bridging was analyzed in HNSCC cell lines and OKF6/TERT-1 cellsgrown on chamber slides (Nalge Nunc International) after treatment withColcemid™ (Irvine Scientific); cells were otherwise untreated. Cellswere arrested in metaphase with 0.1 g ml Colcemid™ for 5 h, washed in1×HBSS, and allowed to resume their respective cell cycles for 3-4 hprior to fixation in Carnoy's fixative (3:1 methanol:glacial aceticacid). Chamber slides were stained with DAPI. A minimum of 500 cellswere analyzed for each cell line, and the frequencies of anaphasebridging and nucleoplasmic bridging were recorded. A test of proportionswas used to compare the frequencies in each of the HNSCC cell linesrelative to the frequency observed in the OKF6/TERT-1 cells (0.74), andp-values were reported.

Chromosome Aberration Analysis.

Flasks of UPCI:SCC084, 104 and 116 were trypsinized and then subjectedto 2.5Gy of IR. The cells were re-seeded into T-25 flasks, and allowedto repair for 48 h prior to being harvested with a final concentrationof 0.1 μg/ml Colcemid™ and 18 nM Calyculin A for 30 min (Calbiochem, SanDiego, Calif.). Since cells lacking a G1 phase cell cycle checkpoint andthose with a defective DNA damage response are often blocked in G2, toavoid skewing of the data, we analyzed the chromosomes from G2 cellsafter premature chromosome condensation with Calyculin A. Harvestedcells were subjected to a hypotonic treatment before being fixation inCarnoy's fixative. Slides were then prepared from the cell pellets anddried overnight, solid stained for 8 min in 4% Giemsa/PBS solution, andrinsed with distilled water. Two researchers independently scored 25cells each per cell line, per treatment. Chromosome aberrationsincluded: chromosome breaks, chromatid gaps or breaks, quadriradials,triradials, rings, dicentrics, and fragments. Chromosome breaks,radials, rings and dicentrics were assigned twice the weight of theother aberrations, as they involve two chromatid breakage events. Thetotal weighted aberrations were summed, and determined per chromosomeand per cell for each treatment. The standard error of the mean was usedas the estimate of error in the sample. A Student's t-test was used tocompare the raw distributions of total weighted aberrations between thecontrol and IR-treated samples.

Clonogenic Survival Assay.

To assess cell survival in response to ionizing radiation, a clonogenicsurvival assay was performed. Two thousand cells were seeded in 60 mmPetri dishes and allowed to attach overnight. Cells were then treatedwith increasing doses of γ-irradiation at 1, 2.5, 5, and 10 Gy. The cellculture medium was changed at the end of seven days. Mock-treated cells,cultured in parallel, were used to determine relative platingefficiency. After 12 days, the colonies were stained with Giemsa (Sigma)and counted. A colony was defined as a cluster of ≧50 cells, presumablyhaving formed from a single cell. All experiments were performed intriplicate, and error is reported as one standard deviation from themean.

7.2 Results

Partial Loss of MRE11A, ATM and H2AFX in HNSCC.

Dual-color FISH was performed with BAC probes to MRE11A, ATM and H2AFXalong with a centromere 11 enumeration probe in HNSCC (CEP 11; D11Z1)(Vysis/Abbott Molecular Inc.). Table 12 summarizes these FISH results.The copy number ratios of the distal 11q genes were determined to beeither lost or normal with respect to CEP. Based on these results, theeleven cell lines were grouped as follows. All cell lines with 11q13amplification were haploinsufficient for MRE11A, ATM and H2AFX,including UPCI:SCC078, 084, 131 and 136 (“Amplification with Loss”). TheHNSCC cell lines, UPCI:SCC104, 142 and 122 were haploinsufficient foreither MRE11A, ATM and/or H2AFX in the absence of 11q13 amplification(“No Amplification with Loss”). UPCI:SCC099, 116 and 182 had neither11q13 amplification nor distal 11q loss (“No Amplification, No Loss”).UPCI:SCC125 is comprised of a highly heterogeneous cell population,making its analysis less straightforward than the other cell lines.There is no evidence for 11q13 amplification in this cell line. Whilethe copy number ratios would suggest that UPCI:SCC125 is normal in copynumber for MRE11A, ATM and H2AFX, FISH results indicated that MRE11A(31% loss, 44% gain), ATM (52% loss, 1% gain), H2AFX (43% loss, 25%gain) gene copy numbers are more a function of the average than thenorm. Thus, for the purposes of this study, this cell line is expectedto respond somewhere between cell lines with “No Amplification withLoss” and those with “No Amplification, No Loss,” given the absence of11q13 amplification.

TABLE 12 Summary of 11 HNSCC Cell Lines, Including FISH Copy NumberResults Expressed as Ratios Relative to the Chromosome 11 Centromere

^(a)Dark shading indicates partial loss or haploinsufficiency.^(b)UPCI:SCC125 is distinguished from the cell lines with “no 11q13 amp,distal 11q loss” because it is heterogeneous with respect to copynumber, as evidenced by the mixture of cells showing gain or loss forany given gene in a single slide preparation.

Confirmation of Physical Loss of Distal 11q was Carried Out by TwoAdditional Methods.

Quantitative microsatellite analysis, or QuMA, was used to map DNA copynumber of sequential microsatellite loci along 11q. The resultingprofiles were grouped according to the FISH data for relative copynumber loss of MRE11A, ATM and H2AFX along 11q. The results, whichdemonstrate reduction in copy number of each of these markers, supportthe findings of our FISH experiments. LOH analysis was performed as anindependent test to validate our FISH and QuMA results, and results wereavailable for nine of the eleven cell lines. The results of LOHanalysis, summarized in FIG. 26 substantiate the loss patterns observedfor distal 11q using FISH and QuMA, as extended segments of 11q wereshown to have complete or partial LOH in the “No Amplification withLoss” and “Amplification with Loss” HNSCC cell line groupings. The celllines with “No Amplification, No Loss” do not have significant LOH along11q. Thus, the results from FISH, QuMA and LOH studies corroborate the11q loss in the HNSCC cell lines.

TABLE 13 Summary of FISH Copy Number Changes in CCND1 and ATM in OvarianTumors, Head and Neck Squamous Cell Carcinomas, and Primary BreastTumors. CCND1 ATM Nor- Nor- Tumor Amp Gain mal Loss Amp Gain mal LossOvarian cancer TP 02-349 N^(a) 0 0 100 0 0 0 99 1 T^(a) 25 11 64 0 0 395 2 TP 02-255 N 0 0 100 0 0 0 100 0 T 4 12 84 0 0 0 85 15 TP 02-217 N 00 100 0 0 2 96 2 T 2 5 93 0 0 0 97 3 TP 02-238 N 0 0 100 0 0 0 100 0 T 48 88 0 0 5 41 54 TP 02-500 N 0 2 98 0 0 2 96 2 T 16 16 66 0 0 5 53 42 TP02-545 N 0 3 97 0 0 2 96 2 T 22 12 66 0 6 10 79 5 TP 02-528 N 0 3 95 2 00 98 2 T 6 12 82 0 0 7 51 42 TP 02-539 N 0 1 97 2 0 0 100 0 T 16 31 53 00 11 87 2 TP 02-505 N 0 0 100 0 0 0 100 0 T 81 14 5 0 0 0 93 7 TP 02-236N 0 2 98 0 0 2 98 0 T 13 17 70 0 89 0 11 0 HNSCC 62102-3 N 0 2 97 1 0 095 5 T 58 23 19 0 0 0 54 46 62009-5 N 1 1 98 0 0 0 97 3 T 11 36 51 2 0 034 66 62134-0 N 0 1 99 0 0 0 100 0 T 98 1 1 0 0 0 47 53 62004-0 N 0 0100 0 0 0 98 2 T 4 0 94 2 0 2 42 56 61936-7 N 0 1 99 0 0 1 98 1 T 99 1 00 0 0 41 59 Breast cancer TP 98-279 T 90 6 4 0 0 0 77 23 TP 98-323 T 062 38 0 0 0 43 57 TP 98-350 T 0 6 94 0 0 1 89 10 TP 98-410 T 0 10 90 0 00 91 9 TP 98-015 T 0 7 91 2 0 2 90 8 TP 99-051 T 91 4 5 0 0 0 70 30 TP99-060 T 0 16 81 3 0 4 88 8 TP 99-084 T 0 5 93 2 0 0 96 4 TP 99-110 T 012 84 4 0 1 88 11 TP 99-117 T 1 7 93 0 0 0 93 7 ^(a)T represents tumortissue and N represents surrounding normal tissue as demarcated byhistological and pathological studies. A minimum of 50 cells werecounted for each tumor and normal tissue studied.

Partial Loss of ATM in Primary Head and Neck, Ovarian and Breast Tumors.

Paraffin FISH was used to study copy number changes in ATM and CCND1 inprimary head and neck squamous cell carcinomas, ovarian and breastcarcinomas. Table 13, above, summarizes these results (see also Section8, below). It was found that three of five primary head and neck tumorsshowed CCND1 amplification, whereas all five primary head and necktumors showed partial loss of ATM compared to CEP 11. Partial ATM losswas also seen in four of ten ovarian tumors studied and three of tenbreast carcinomas studied. For the primary head and neck and ovariantumors, the changes in CCND1 and ATM copy number were also studied inthe tumor adjoining normal tissue. Thus, these results indicate thatpartial loss of ATM is associated with tumor types in addition to HNSCC.

Distal 11q Loss is Associated with Chances in Expression of MRE11A, ATMand H2AFX.

Taqman quantitative reverse transcriptase PCR (RT-PCR) andimmunoblotting were performed to assess whether loss of MRE11A, ATM andH2AFX led to a reduction in their expression (FIG. 29A). The detectableexpression of ATM and H2AX in the HNSCC generally correlated with genecopy number, especially for the ATM gene. The “Amplification with Loss”cell lines are haploinsufficient based on physical loss of distal loci,and generally exhibit reduced RNA expression relative to controls. SCCHNcell lines are heterogenous for loss of distal 11q and not all cellswithin a cell line show this loss. This heterogeneity is reflected inthe relative RNA and protein expression in our studies.

Protein expression in HNSCC cell lines and various controls was assessedby immunoblotting (FIG. 29B). The results show that cell lines withdistal 11q loss tend to have an overall lower expression level of MRE11,ATM and H2AX in comparison to those cell lines without distal 11q loss.The relative protein expression data correlate with the quantitativeRT-PCR analysis. The results also show that untreated HNSCC cell lineshave low levels of γ-H2AX, and nearly uniform increases in H2AXphosphorylation one hour after treatment with 2.5 Gy IR. These resultsare not particularly correlated with γ-H2AX focus formation, which is awidely accepted measure of the DNA damage response. Changes in H2AXprotein phosphorylation may not be detectable by immunoblotting in ourHNSCC cell lines.

Decreased Expression of MRE11A, ATM and H2AFX Correlates with Aberrantγ-H2AX Foci.

γ-H2AX focus formation was evaluated after treatment with 2.5 Gy IR.Constitutive γ-H2AX foci were seen in some of the cell lines(UPCI:SCC099 and 116, among others) in the absence of exogenous geneticinsult. Hence, γ-H2AX focus formation following IR was assayed byseveral measurements, including the mean number of foci per cell, thedistribution of foci in 200 cells, and the percent of focus-positivecells. Comparison of the mean number of foci per cell in mock-treatedand IR-exposed cells is shown in FIG. 27. The highest mean numbers offoci were seen in the cell lines “No Amplification, No Loss”(min=5.93±0.75; max=8.20±1.16), and these were similar to those of thecontrol fibroblast cell line (10.34±0.65). The cell lines with“Amplification with Loss” showed the lowest mean levels of γ-H2AX foci.When treated with IR, the mean number of γ-H2AX foci was more thantwo-fold lower in the cell lines with amplification and loss than celllines without 11q loss (min=3.93±1.23; max=6.05±0.87). “No Amplificationwith Loss” cell lines also showed low levels of γ-H2AX focus formationafter IR (min=2.38±1.46; max=7.81±1.03). While there was γ-H2AX focusformation in the mock-treated ATM−/− control cell line, there wasessentially no difference between the mock-treated and IR-treated cellsafter one hour, thereby serving as a negative control. Cell lines thathave fewer focus-positive cells and have a reduced mean number of fociper cell following IR are considered defective in γ-H2AX focusformation. The distribution of focus-positive cells also reveals adeficient response when the data are categorized as 0-2, 3-10 and >10foci (FIG. 30A-B). Following IR, the distribution of focus-positivecells with “No Amplification, No Loss” closely resembles thedistribution seen in normal fibroblasts. The “Amplification with Loss”and “No Amplification with Loss” cell lines tend to show more deficientresponses than the “No Amplification, No Loss” or normal fibroblasts, assmall proportions of these cells have more than 10 foci. The signaldistribution in the ATM null cell line is essentially unchangedfollowing IR.

Since H2AX phosphorylation and formation of γ-H2AX foci occurs inresponse to double strand breaks or apoptotic DNA fragmentation,late-appearing foci may represent irreparable damage to the cell(Rogakou et al., 2000). To evaluate if the γ-H2AX foci we observed werein response to double strand breaks or as a precursor to apoptosis, acell viability assay was performed. Significant cell death was notexpected to occur within 1 h as a result of a 2.5 Gy IR treatment. InUPCI:SCC116, 94.0±0.05% of the control population and 92.6±0.15% of thetreated cells remained viable (FIG. 31). In UPCI:SCC084, 90.7±0.07% ofthe control population and 91.7±0.09% of the treated population wereviable. In UPCI:SCC104, 94.1±0.04% of the control cells and 91.1±0.08%of the treated cells remained viable. As expected, the control andtreated confidence intervals overlap in all three cases, indicating thatthere was no difference between the cell lines with respect to survivaland confirming that cell death did not significantly skew the focusformation assay results.

Loss of Distal 11q is Associated with Increased Chromosomal Instability.

The number of weighted aberrations per chromosome 48 h after IR wasdetermined for UPCI:SCC084, 104 and 116 by using Calyculin A toprematurely condense chromatin for analysis of G2 and mitotic phasechromosomes (FIG. 7D and Table 14A). Breaks were evident in the celllines with loss of distal 11q, irrespective of 11q13 status. Completeresults are summarized in Table 13a. There is no significant differencein the total chromosomal aberrations per cell between the control(2.52±0.97%) and IR-treated (2.94±0.62%) populations in UPCI:SCC116,which is from the “No Amplification, No Loss” grouping. There issubstantial agreement between the results for UPCI:SCC084 (C=1.43±0.51%;IR=6.68±1.40%; p<0.001) and UPCI:SCC104 (C=3.15±0.76%; IR=6.68±1.05%,p<0.001), both of which have loss of distal 11q. Thus, the cell lineswith distal 11q loss, regardless of amplification status (UPCI:SCC084(Amplification with Loss) and UPCI:SCC104 (Loss, No Amplification))demonstrated significantly higher levels of chromosomal aberrations inresponse to ionizing radiation when compared to a cell line withoutdistal 11q loss (UPCI:SCC116; No Amplification, No Loss).

The frequency of anaphase bridges in 500 cells was used an indicator ofchromosomal instability. The frequency of anaphase bridges in each cellline is summarized as a percentage in Table 14B. Overall, the lowestfrequency was seen in OKF6/TERT-1 cells (0.74%). The lowest frequencyamong all HNSCC cell lines was seen in UPCI:SCC116 (0.96%), and thehighest frequency was seen in UPCI:SCC142 (5.63%). In cell lines without11q loss, the maximum frequency (1.31%; UPCI:SCC099) was lower than anyof the frequencies observed in cell lines with 11q loss (in the“Amplification with Loss” and “Loss, No Amplification” groups). In acomparison of proportions, all cell lines with distal 11q loss,irrespective of 11q13 amplification, have significantly elevatedfrequencies of anaphase bridges relative to the immortalized oralkeratinocytes and “No Amplification, No Loss” cell lines. For two of thecell lines, UPCI:SCC078 and 122, the p-value is less than 0.1. For allother cell lines with distal 11q loss, the p-value is less than 0.05.Since the frequency of anaphase bridges was measured in untreated celllines, the intrinsic level of chromosomal instability is higher in celllines with 11q loss.

TABLE 14A Results from the Chromosomal Aberration Assay forRepresentative Cell Lines

^(a)Dark shading indicates a significant result for the Student's t-test(p<0.001).

TABLE 14B Frequency of Anaphase Bridges in Untreated HNSCC Cell Lines

^(a)Test of proportions to compare the frequency of anaphase bridges ineach cell line to the frequency observed in immortalized oralkeratinocytes (0.74%). Shading indicates a significant difference; lightgray shading denotes p<0.1, and dark gray shading indicates values wherep<0.05.

TABLE 15 Dose of Individual Grouped Cell Line IR Gy) Mean ± SD Mean ± SDNHOK 0 100.0 ± 0.0  1 59.7 ± 2.1 2.5 30.3 ± 0.6 5  6.7 ± 0.6 10  0.0 ±0.0 UPCI:SCC SCC099 0 100.0 ± 0.0  1 58.0 ± 1.0 2.5 29.7 ± 1.5 5  5.7 ±1.2 10  0.0 ± 0.0 SCC116 0  100 ± 0.0 1 55.0 ± 4.4 2.5 23.0 ± 2.6 5  6.0± 1.0 10  0.0 ± 0.0 SCC182 0  100 ± 0.0 1 56.0 ± 5.2 2.5 32.7 ± 4.0 5 9.3 ± 2.1 10  0.0 ± 0.0 No Amp No Loss 100.0 ± 0.0  56.3 ± 3.7 28.4 ±5.0  7.0 ± 2.2  0.0 ± 0.0 SCC078 0  100 ± 0.0 1 83.0 ± 2.6 2.5 56.7 ±0.6 5 38.0 ± 1.7 10  7.0 ± 0.0 SCC084 0  100 ± 0.0 1 80.7 ± 4.6 2.5 52.3± 5.0 5 24.7 ± 2.1 10  7.7 ± 0.6 SCC131 0  100 ± 0.0 1 86.7 ± 2.5 2.551.7 ± 3.8 5 37.3 ± 2.1 10 11.3 ± 2.5 SCC136 0  100 ± 0.0 1 81.7 ± 3.52.5 55.0 ± 4.6 5 34.7 ± 2.5 10 11.0 ± 1.0 Amp with Loss 100.0 ± 0.0 83.0 ± 3.8 53.9 ± 3.9 33.7 ± 5.9  9.3 ± 2.3 SCC104 0  100 ± 0.0 1 82.3 ±4.7 2.5 55.3 ± 2.1 5 33.7 ± 1.2 10  7.7 ± 0.6 SCC122 0  100 ± 0.0 1 77.0± 1.0 2.5 54.7 ± 0.6 5 24.0 ± 1.7 10  4.0 ± 1.0 SCC142 0  100 ± 0.0 181.0 ± 4.0 2.5 51.7 ± 4.6 5 35.3 ± 1.5 10  9.7 ± 0.6 No Amp with Loss100.0 ± 0.0  80.1 ± 4.0 53.9 ± 3.1 31.0 ± 5.5  7.1 ± 2.6 SCC125 0  100 ±0.0 1 83.3 ± 5.0 2.5 40.3 ± 3.2 5 27.7 ± 0.6 10  0.7 ± 0.6

Loss of Distal 11q in HNSCC is Associated with Reduced Sensitivity to IR(Radiosurvival).

A clonogenic survival assay was used to determine the sensitivity ofHNSCC to DNA damage induced by ionizing radiation (FIG. 28A-B, see alsoTable 15, above). Results for the assay in triplicate were grouped as“No Amplification No Loss” (UPCI:SCC099, 116 and 182), “Amplificationwith Loss” (UPCI:SCC078, 084, 131 and 136), and “No Amplification withLoss” (UPCI:SCC104, 122 and 142). Normal human oral keratinocytes (NHOK)were included as a control. UPCI:SCC125 was not included in the groupanalysis due to the heterogeneity in copy number revealed by FISH. Inthe grouped analysis, NHOK showed 59.7±2.1% survival at 1 Gy, and nosurvival at 10 Gy of IR. Similarly, the HNSCC cells with “NoAmplification, No Loss” showed 56.3±3.7% survival at 1 Gy, and nosurvival at 10 Gy IR. Thus, the normal human oral keratinocytes (NHOK)and cell lines without 11q loss are sensitive to ionizing radiation.Conversely, the “Amplification with Loss” HNSCC cell lines had 83±3.8%survival at 1 Gy, and 9.3±2.3% survival at 10 Gy IR. The “NoAmplification with Loss” cells had 80.1±4.0% survival at 1 Gy, and7.1±2.6% survival after exposure to 10 Gy IR. The UPCI:SCC125 cells hada high surviving fraction after 1 Gy (83.3±5.0%), and a small, butvisible surviving fraction at 10 Gy (0.7±0.6%). Thus, unexpectedly, theHNSCC cell lines with distal 11q loss (“Amplification and Loss”(UPCI:SCC078, 084, 131 and 136) and “No Amplification with Loss”(UPCI:SCC 104, 122 and 142)) demonstrate loss of sensitivity to ionizingradiation with a substantial proportion of cells surviving a usuallylethal single fraction of 10 Gy IR (radiosurvival). The termradiosurvival is used to refer to the observation that 10% of HNSCCcells with 11q loss survive the dose of 10 Gy IR, whereas none of thecontrol cells or HNSCC cells without 11q loss survive the same dose(Golding et al., 2004).

7.3 Discussion

Amplification of 11q13 occurs in about 45% of HNSCC and is associatedwith a poor prognosis. Previously, Roh and colleagues determined that11q13 amplification was an early event in HNSCC development, at timesappearing in dysplastic tissue (Roh et al., 2000). In the BFB cyclemodel of 11q13 amplification, distal 11q loss necessarily precedes 11q13amplification and is therefore an earlier event in HNSCC tumorigenesis.While the amplification of genes in band 11q13 is thought to play animportant role in the progression of HNSCC, the impact of the preceedingloss of genes on distal 11q has not been examined in detail. In additionto HNSCC, loss of distal 11q has been reported in a variety of tumorsincluding breast carcinoma, esophageal carcinoma, cervical carcinoma andprostate cancer (Tomlinson et al., 1995; Dahiya et al., 1997; Evans etal., 1998; Jin et al., 1998, 2006; Matsumoto et al., 2004; Miyai et al.,2004; Wang et al., 2004). Genes encoding several proteins critical tothe DNA damage response, including MRE11A, ATM and H2AFX, are located ondistal 11q. Thus, loss of distal 11q may lead to defects in DNA damagerecognition and repair and play a role in chromosomal instability andaltered response to therapy in a variety of tumors.

The data presented herein confirms the loss of distal 11q in a largeproportion of HNSCC by FISH (Table 12), LOH (FIG. 26) and QuMA. Loss ofdistal 11q in HNSCC was also shown by Jin et al. (1998, 2006). While LOHand QuMA showed segmental loss from approximately 11q14.2 to 11qter,FISH analysis directly demonstrated relative copy number loss of MRE11A,ATM, and H2AFX in a subset of our HNSCC cell lines. In addition to HNSCCcell lines, we observed relative loss of ATM copy number in all fiveprimary head and neck tumors studied. Although at a lower frequency, ATMloss was also observed in at least 60% of a small, but unselected sampleof ovarian and breast carcinomas.

In response to ionizing radiation, a reduction in γ-H2AX focus formationwas observed in HNSCC cell lines with distal 11q loss irrespective of11q13 amplification. These cell lines had considerably reduced meannumbers of foci after treatment with IR compared with normal controls orHNSCC cell lines without loss of distal 11q. This could result fromeither a reduction in H2AX protein present in the chromatin around DSBs,a reduction in the ATM kinase activity that phosphorylates H2AX afterDNA damage or the disruption of substrate-kinase interactions. SinceH2AX phosphorylation following IR is partially ATM-dependent, it ispossible that the reduction in ATM protein, and perhaps concomitantreduction in activity, is responsible for the reduced number of foci.Given that γ-H2AX focus formation has been shown to be a validassessment of continuity of elements in the DNA damage response (Rogakouet al., 1998; Paull et al., 2000), particularly those involved upstreamin the recognition of strand breaks, the results above indicate thatdistal 11q loss is associated with a compromised DNA damage response.

Chromosomal breakage was examined following treatment with IR as ameasure of chromosomal instability in HNSCC, and anaphase bridgeformation in untreated HNSCC was calculated as a measure of baselinechromosomal instability. As shown in FIG. 7A-D and Table 14A,UPCI:SCC084 (Amplification with Loss) and UPCI:SCC104 (No Amplificationwith Loss) demonstrate a significant increase in the number ofchromosome aberrations following IR. In comparison, UPCI:SCC116, whichhad no amplification or distal loss, did not demonstrate a significantincrease in IR-dependent chromosomal aberrations. Thus, elevatedchromosomal breakage levels correlate with 11q loss, partialinsufficiency for MRE11A, ATM and H2AFX, and deficits in γ-H2AX focusformation. Anaphase bridges were found to be increased in the subset ofcell lines with loss of distal 11q, regardless of 11q13 amplification(Table 14B). Cell lines without loss of distal 11q had frequenciessimilar to those of normal cells. In addition to being a hallmark ofchromosomal instability, an increased incidence of anaphase bridgessupports the BFB mechanism for gene amplification. It has been shownthat mre11 and tefu (dATM) knockout mutants in Drosophila melanogasterexhibit a high frequency of telomere fusions, dicentric chromosomes andanaphase bridging (Bi et al., 2004; Ciapponi et al., 2004). TheDrosophila dicentric chromosomes break and initiatebreakage-fusion-bridge cycles, similar to the anaphase bridges and BFBcycles seen in HNSCC with distal 11q loss. Partial loss of MRE11A andATM, as well as other genes, is thought to result from the deletionevent that can precede BFB cycles, eventually leading to 11q13amplification in HNSCC. Additional consequences for the cell result fromamplification of CCND1 and the other genes in the 11q13 amplicon,including FADD, TAOS1, TAOS2, and EMS1 (Huang et al., 2002; Huang etal., 2006). The observations of BFB cycles as the primary mechanism of11q13 amplification (Reshmi et al., 2007), as well as the observation ofincreased chromosomal instability in cell lines with 11q loss are strongevidence that loss of distal 11q is an important factor that precedes11q13 gene amplification and may play a role in the development and/orprogression of HNSCC.

Survival of the HNSCC cell lines following IR was investigated using aclonogenic survival assay. Since chromosome instability and deficits inγ-H2AX focus formation were observed following treatment with IR inHNSCC cell lines with 11q deletions, it had been expected that thesecell lines would also be highly sensitive to ionizing radiation. Indeed,previous studies have shown that loss of ATM or MRE11A correlates withradiosensitivity (Gatti et al., 1991; Stewart et al., 1999; Prime etal., 2001). Surprisingly, it was found that loss of distal 11q in HNSCCis associated with radiosurvival, as 7-10% of cells in all tested HNSCCcell lines with distal 11q loss survived 10 Gy IR in a single fraction,whereas none of the control cells, including HNSCC cells without 11qloss, survived this dose of IR. Thus, counterintuitively, distal 11qloss appears to be associated with both chromosomal instability andradiosurvival. Mutations in TP53 between exons 5-9 are summarized inTable 12. Since there was equal representation of wild-type and mutantTP53 in all three groups of cell lines, TP53 alterations should not beresponsible for the observed radiosurvival in the “Loss, NoAmplification” and “Amplification with Loss” cells. Radiosurvival doesnot appear to be associated with loss of any specific gene on distal11q, but there is a possibility that other undocumented differences inthe cell lines may contribute to the observed deficiencies in the DNAdamage response and the radiosurvival. Previous studies have postulatedthat inhibition of ATM kinase activity in tumors may increase theefficacy of radiotherapy and genotoxic chemotherapy in the treatment ofTP53-mutant cancers (Westphal et al., 1998; Fedier et al., 2003).Without making any claims as to the nature of the TP53 mutations in thecell lines (Table 12), the evidence indicates that HNSCC cell lines,regardless of the presence of functional TP53, do not necessarilyrespond uniformly to ionizing radiation. The findings herein show thatdespite IR-dependent chromosomal instability and deficits in γ-H2AXfocus formation, the HNSCC cell lines with 11q loss exhibitradiosurvival. This suggests that increased chromosomal damage inresponse to ionizing radiation may not predict radiosensitivity, andquestions the efficacy of using an ATM-inhibitor in the treatment ofHNSCC. The association between distal 11q loss and radiosurvival in celllines may have direct translational implications for HNSCC patients, asaggressive radiation therapy may not be beneficial to patients withdistal 11q loss.

Recently, a number of studies have shown that the DNA damage responseprotects against tumor formation, and that inactivation of components ofthe DNA damage response pathway may lead to chromosomal instability,uncontrolled cell proliferation and tumorigenesis (Bartkova et al.,2005; Gorgoulis et al., 2005). The data herein indicate that, along withCCND1 amplification, distal 11q loss is an early event in HNSCCtumorigenesis and promotes chromosomal instability. Since a number ofHNSCC cell lines and the tumors from which they were derived arehaploinsufficient for distal 11q, the remaining allele of MRE11A, ATM,H2AFX or other genes on distal 11q could undergo mutation or deletionleading to complete loss of function of the particular gene.Alternatively, it is possible that in a carrier of an ATM mutation, thenormal allele could be deleted during tumor formation, resulting inabsence of a functional allele. The data presented herein indicatesthat, following loss of distal 11q, the pathways that rely on theMRE11A, ATM, and H2AFX genes for DNA damage recognition and repair arecompromised, and no longer function sufficiently to maintain chromosomalintegrity. This loss of the normal DNA damage response appears topromote chromosomal instability and foster a genetic environment thatselects for tumor cells with a growth advantage. Thus, loss of distal11q in HNSCC appears to lead to chromosomal instability and contributesto tumor development, progression, and resistance to therapy.

8. EXAMPLE Loss of Distal 11q in Carcinomas from Multiple Sites

8.1 Materials and Methods

Cell Culture.

HNSCC cell lines, normal human oral keratinocytes (NHOK), and normalhuman fibroblasts were established in the inventors' laboratory.hTERT-transfected human oral keratinocytes were OKF6/TERT-1 from thelaboratory of Dr. James G. Rheinwald; Dickson et al., 2000. GM09607, anATM null cell line, was from Coriell Cell Repositories, Camden, N.J. TheHNSCC cell lines were grown in Minimal Essential Medium (GibcoInvitrogen, Grand Island, N.Y.), supplemented with 1% non-essentialamino acids, 1% L-glutamine, 0.05 mg/ml Gentamicin and 10% FBS) (allsupplements from Gibco Invitrogen) (White, et al. 2007). NHOKestablished from uvulopalatopharyngoplasty specimens were cultured inserum-free KGM-2 medium (Clonetics, Walkersville, Md.), supplementedwith BPE (bovine pituitary extract), hEGF, insulin (bovine),hydrocortisone, GA-1000 (Gentamicin, Amphotericin B), epinephrine andtransferrin as per the manufacturer's instructions (supplements suppliedin the KGM-2 BulletKit™ from Clonetics). Finite passage normal humanfibroblasts established from uvulopalatopharyngoplasty tissue werecultured in Minimal Essential Medium (Gibco Invitrogen), supplementedwith 1% non-essential amino acids, 1% L-glutamine, 0.05 mg/ml Gentamicinand 10% FBS) (Gibco Invitrogen). The OKF6/TERT-1 cells were initiallycultured in Keratinocyte-SFM supplemented with 25 μg/ml bovine pituitaryextract, 0.2 ng/ml epidermal growth factor, 0.3 mM CaCl2 andpenicillin-streptomycin (Gibco Invitrogen). These keratinocytes wereexpanded to high density in a 1:1 mixture of Keratinocyte-SFM andDMEM-F12. The DMEM-F12 was a 1:1 mixture of calcium-free andglutamine-free DMEM and Ham's F-12 supplemented with 25 μg/ml bovinepituitary extract, 0.2 ng/ml epidermal growth factor, 1.5 mM L-glutamineand penicillin-streptomycin (Gibco Invitrogen). GM09607 (Coriell CellRepositories, Camden, N.J.) was cultured in Dulbecco's Modified EagleMedium (DMEM), supplemented with 1% non-essential amino acids, 0.05mg/ml penicillin-streptomycin-L-glutamine, and 10% FBS (GibcoInvitrogen). Breast, lung, ovarian, and prostate cancer and multiplemyeloma cell lines were obtained from various sources and grown underculture conditions recommended by the originator.

Fluorescence In Situ Hybridization (FISH).

To prepare mitotic cells for FISH analyses, carcinoma cells wereharvested following a 5 h treatment with 0.1 μg/ml Colcemid™(IrvineScientific, Santa Ana, Calif.), hypotonic KCl (0.075M) treatment for 16min, and fixation in 3:1 methanol:glacial acetic acid. All other cellswere harvested using the same method, except that 1 h of Colcemid™ waspreferred for non-tumor cells. FISH analysis was used to detect copynumber changes in the CCND1 and ATM genes in the cancer cell lines. ForFISH analysis, cells were harvested, dropped onto slides, treated withRNase/2×SSC, and dehydrated using a graded series (70%, 80% and 100%) ofethanol washes. Chromatin was denatured with 70% formamide and the cellswere dehydrated in a second graded series of ethanol washes. The CCND1and ATM probes for FISH were prepared following DNA extraction from BACspurchased from Children's Hospital of Oakland Research Institute (CHORI,Oakland, Calif.). The BAC DNA was isolated and labeled using a nicktranslation kit from Vysis/Abbott Molecular Inc. (Des Plaines, Ill.).The labeled DNA was precipitated with ethanol, resuspended inhybridization buffer, denatured for 5 min at 75° C., and preannealed for15-30 min at 37° C. Each probe was hybridized for 16 h at 37° C., afterwhich slides were washed with SSC/Tween-20. Slides were counterstainedwith DAPI and mounted with antifade prior to analysis. All FISH analyseswere carried out using an Olympus BX-61 epifluorescence microscope(Olympus Microscopes, Melville, N.Y.). An Applied Imaging CytoVisionworkstation with Genus v3.6 software was used for image capture andanalysis (Applied Imaging, San Jose, Calif.).

Paraffin and Frozen Section FISH.

Anonymous frozen sections of breast carcinomas and paraffin sections ofthe stage III ovarian carcinomas were obtained from the Magee-WomensHospital Tissue Procurement Program. The paraffin sections of anonymousHNSCC were obtained from the Head and Neck SPORE Tissue Bank at theUniversity of Pittsburgh. 4-5 μM thick sections of formalin-fixed,paraffin-embedded tissue were mounted on positively charged microscopeslides. The slides were aged overnight at 60° C., following which theywere deparaffinized twice with xylene for 5 min each at roomtemperature. The slides were dehydrated in two 100% ethanol washes, 2min each at room temperature. The slides were then treated with0.5×SkipDewax solution (Insitus Biotechnologies, Albuquerque, N. Mex.)at 80° C. for 15 min followed by a wash in distilled water for one minat room temperature, treatment in 0.2N HCl for 20 min at roomtemperature, pretreatment with 1 M sodium thiocyanate at 80° C. for 30min, a wash in distilled water for one min at room temperature, twowashes with 2×SSC 5 min each, and then treatment with protease (pepsin)solution for 15 min at 37° C. Next, the slides were fixed in 10%Formalin for 10 min at room temperature. After two 2×SSC washes at roomtemperature, the slides were dehydrated with a graded series of ethanolwashes at room temperature, and allowed to dry on slide warmer. Next,slides were denatured at 75° C. in 70% formamide, followed by a coldseries of ethanol washes. The probes for paraffin FISH were prepared asdescribed for FISH above. The hybridization of the FISH probes andpost-hybridization treatment of the paraffin sections was carried out asdescribed for FISH above. 4-5 μM thick sections of OCT-embedded, freshfrozen breast carcinomas were mounted on positively charged microscopeslides. The slides were placed in 0.8% sodium citrate for 20 min, fixedin 3:1 methanol acetic acid, and air dried. The slides were aged at 90°C. for 10 min, digested in 0.005% pepsin in 0.2N HCl at 37° C. for 60sec, incubated in 70% ethanol for 30 sec, pretreated in 2×SSC at 37° C.for 60 min, dehydrated in a graded series of ethanol, denatured andhybridized as for FISH above.

8.2 Results and Discussion

Dual-color FISH with BAC probes to CCND1 and ATM were performed alongwith a centromere 11 enumeration probe in HNSCC (CEP 11; D11Z1)(Vysis/Abbott Molecular Inc.). Table 16, below, summarizes the FISHresults. The copy number ratio of the ATM gene and the CCND1 gene wasdetermined to be lost, gained, amplified or normal with respect to thecentromere enumeration probe for chromosome 11. Arbitrary cut-off valuesof 15% were used for ATM loss and 10% for CCND1 amplification inparaffin sections.

The results show that distal 11q, marked by ATM is lost in 19 of 26(73%) primary HNSCC examined and that CCND1 is amplified in 8 of 26(31%) primary HNSCC. Primary ovarian tumors, reported first by Parikh etal. (Parikh, et al. 2007) showed loss of distal 11q in 2/10 (20%) andcyclin D1 amplification in 60%. Primary breast tumors showed loss in29/68 (43%) and cyclin D1 amplification in 10/68 (15%). Loss of distal11q with or without 11q13 amplification is seen in cell lines derivedfrom HNSCC, ovarian, breast, prostate, and lung cancer cell lines andmultiple myeloma cell lines.

The literature shows that cancer patients with ATM gene loss have aworse prognosis than those without ATM gene loss. Further, a recentstudy showed that the residual ATM allele is mutated in 36% of B-cellchronic lymphocytic leukemias (CLL) with a distal 11q deletion, and thatthese CLLs express a defective cellular response to ionizing radiationor cytotoxic drug exposure in vitro (Austen, et al. 2007). Further,these authors found that inactivation of the second ATM allele wasassociated with decreased survival beyond that already associated by an11q deletion. In an immunohistochemistry study of the predictive valueof molecular markers in squamous cell carcinoma of the esophagus, theauthors found that tumors positive for ATM kinase phosphorylated atSer1981 and CHK2 kinase phosphorylated at Thr68 (markers that werecorrelated with each other) responded to radiochemotherapy (45 Gy IR,cisplatin and 5-fluorouracil) with tumor regression and had betteroverall survival compared to tumors that were negative for CHK2expression (Sarbia, et al. 2007). Another recent study of ATM expressionin 70 gastric cancer specimens showed consistent decreased expressionand phosphorylation of the protein and revealed that a low level ofphosphorylated ATM was statistically significantly correlated with poordifferentiation, lymph node metastasis, and poor 5-year survival (Kang,et al. 2008). Analysis of ATM expression in breast cancer showed thatATM expression levels in tumors were decreased by approximately 50%compared to adjacent normal mucosa (Ye, et al. 2007). Further, patientswith the lowest tertile of ATM mRNA had less favorable disease-freesurvival and overall survival compared to patients in the upper twotertiles (Ye, et al. 2007). In a study that shows the importance of 11qloss in breast cancer, the protein expression of ATM and theMRE11/RAD50/NBS1 complex were examined by immunohistochemistry in tumorsfrom 224 women with early breast cancer who were randomized to receiveadjuvant chemotherapy or postoperative radiotherapy (Soderlund, et al.2007). The authors found that the staining intensity of these fourproteins was decreased in the majority of tumors compared to thestaining of normal breast tissue. Weak expression of the MRN complexcorrelated with high histologic grade and estrogen receptor negativity.Importantly, the greatest benefit of radiotherapy was seen in patientswith moderate/strong expression of the MRN complex, whereas patientswith negative/weak MRN expression had no benefit of radiotherapycompared with adjuvant chemotherapy, suggesting that an intact MRNcomplex is critical to the tumor-cell killing effect of radiotherapy.These results all support the idea that the diminished DNA damageresponse resulting from distal 11q loss is associated with reducedsensitivity to IR and a worse prognosis in cancer patients.

A number of studies have shown that the DNA damage response protectsagainst tumor formation, and that inactivation of components of the DNAdamage response pathway may lead to chromosomal instability,uncontrolled cell proliferation and tumorigenesis (Bartkova, et al.2005; Gorgoulis, et al. 2005). The results presented herein indicatethat along with CCND1 amplification, distal 11q loss is an early eventin tumorigenesis and promotes chromosomal instability. Since a number ofcancer cell lines and the tumors from which they were derived arehaploinsufficient for distal 11q, the remaining allele of MRE11A, ATM,H2AFX or other genes on distal 11q could undergo mutation or deletionleading to complete loss of function of the particular gene.Alternatively, it is possible that in a carrier of an ATM mutation, thenormal allele could be deleted during tumor formation, resulting inabsence of a functional allele. It has been demonstrated that, followingloss of distal 11q, the pathways that rely on the MRE11A, ATM, and H2AFXgenes for DNA damage recognition and repair are compromised, and nolonger function sufficiently to maintain chromosomal integrity. Thisloss of the normal DNA damage response appears to promote chromosomalinstability and foster a genetic environment that selects for tumorcells with a growth advantage. Thus, loss of distal 11q in cancer cellsappears to lead to chromosomal instability and contributes to tumordevelopment, progression, and resistance to therapy.

TABLE 16 Copy number alterations in CCND1 and ATM in tumors and cancercell lines from various sites. FISH CCND1 ATM Percentage of cells witheach copy number alteraiton Specimen Type Sample ID Loss Normal Gain AmpLoss Normal Gain Amp Breast cancer T47-D 1 81 18 0 4 91 5 0 cell linesMCF-7 1 50 49 0 93 7 0 0 Multiple Myeloma RPMI 34 52 14 0 17 60 22 1OPM2 0 62 38 0 42 54 4 0 U266 89 11 0 0 73 25 2 0 H929 84 15 1 0 77 2077 0 Lung cancer 54T 0 6 81 12 4 96 0 0 cell lines 84T 0 0 83 16 87 11 00 94T 0 1 81 17 6 92 2 0 98T 0 73 27 0 22 78 0 0 201T 0 59 41 0 13 87 00 253T 0 78 22 0 93 7 0 0 273T 0 94 6 0 35 65 0 0 343T 0 88 9 3 0 25 750 Ovarian cancer OVCAR-3 27 55 17 0 3 97 0 0 cell lines ES-2 0 0 32 6895 5 0 0 SKOV-3 4 89 6 0 100 0 0 0 Ovarian Primary TP02-349 1A 0 64 1125 2 95 3 0 tumors TP02-255 1E 0 84 12 4 15 85 0 0 TP02-217 D 0 93 5 2 397 0 0 TP02-238 2D 0 88 8 4 54 41 5 0 TP02-500 7A 0 68 16 16 0 53 5 42TP02-545 1B 0 66 12 22 5 79 10 6 TP02-628 3C 0 82 12 6 42 51 7 0TP02-539 1G 0 53 31 16 2 87 11 0 TP02-505 2C 0 5 14 81 7 93 0 0 TP02-2362B 0 70 17 13 0 11 0 89 Prostate tumor 1532T 0 1 66 33 97 1 2 0 celllines 1542T 1 45 54 0 0 75 25 0 PC-3 2 96 2 0 15 85 0 0 PCC1 100 0 0 0100 0 0 0 DU145 1 97 2 0 0 96 4 0 LAPC4 17 80 3 0 74 26 0 0 LNCap 6 92 20 12 88 0 0 Breast Primary TP98-2 3 89 8 0 7 93 0 0 tumors in OCTTP98-15 2 91 7 0 8 90 2 0 TP98-63 3 92 5 0 9 89 2 0 TP98-84 0 91 9 0 789 4 0 TP98-98 0 72 26 2 8 92 0 0 TP98-102 1 94 5 0 4 95 1 0 TP98-130 091 9 0 6 94 0 0 TP98-146 0 79 21 0 17 82 1 0 TP98-155 0 89 11 0 18 81 10 TP98-167 3 94 3 0 6 94 0 0 TP98-175 0 93 7 0 8 92 0 0 TP98-176 11 6123 5 48 52 0 0 TP98-182 0 42 12 47 18 80 2 0 TP98-206 0 98 2 0 5 95 0 0TP98-221 0 34 0 66 51 48 1 0 TP98-230 3 95 2 0 32 64 4 0 TP98-237 9 84 70 12 86 2 0 TP98-258 11 80 9 0 1 97 1 1 TP98-268 2 95 3 0 4 94 2 0TP98-276 36 64 0 0 49 51 0 0 TP98-279 0 4 6 90 23 77 0 0 TP98-280 0 94 60 8 91 1 0 TP98-290 13 70 15 2 18 82 0 0 TP98-298 17 73 8 2 10 87 3 0TP98-299 2 79 18 2 7 91 2 0 TP98-310 2 22 17 59 44 55 1 0 TP98-322 0 169 75 24 76 0 0 TP98-323 0 38 62 0 57 43 0 0 TP98-324 11 89 0 0 1 93 6 0TP98-334 0 90 10 0 7 93 0 0 TP98-342 0 91 9 0 5 95 0 0 TP98-350 0 94 6 010 89 1 0 TP98-355 0 97 3 0 6 94 0 0 TP98-376 3 89 8 0 5 93 2 0 TP98-3910 93 7 0 10 90 0 0 TP98-410 0 90 10 0 9 91 0 0 TP98-411 1 88 11 0 6 94 00 TP98-412 0 87 13 0 23 76 1 0 TP98-418 1 94 5 0 8 92 0 0 TP99-31 3 8611 0 9 91 0 0 TP99-33 0 93 7 0 8 92 0 0 TP99-36 0 4 2 94 22 77 1 0TP99-39 0 89 11 0 9 90 1 0 TP99-46 1 90 9 0 5 95 0 0 TP99-51 0 5 4 91 3070 0 0 TP99-52 8 80 12 0 6 86 8 0 TP99-60 3 81 16 0 8 88 4 0 TP99-65 082 18 0 10 90 0 0 TP99-71 0 31 69 0 46 54 0 0 TP99-74 0 86 14 0 9 90 0 0TP99-83 4 79 17 0 18 80 2 0 TP99-84 2 93 5 0 4 96 0 0 TP99-85 15 71 13 025 73 2 0 TP99-88 18 43 35 4 24 76 0 0 TP99-89 11 78 11 0 41 52 7 0TP99-96 17 67 17 0 6 86 9 0 TP99-98 2 15 54 3 65 35 0 0 TP99-99 38 50 112 14 84 2 0 TP99-106 0 4 30 65 72 28 0 0 TP99-109 16 64 20 0 22 65 12 0TP99-110 4 84 12 0 11 88 1 0 TP99-111 0 0 4 96 16 84 0 0 TP99-117 0 93 70 7 93 0 0 TP99-118 12 86 0 0 62 38 0 0 TP99-130 5 87 8 0 46 52 0 1TP99-134 0 50 25 25 54 45 1 0 TP99-137 0 95 5 0 38 62 0 0 TP99-146 0 8119 0 22 78 0 0 HNSCC Paraffin 62097-4 1F 2.0 58.0 31.0 9.0 28 72 0 0Primary tumors 62139-5 3G 2.0 62.0 22.0 14.0 58 40 2 0 62141-8 C 2.083.0 13.0 2.0 5 95 0 0 62165-4 3K 0 73 23 4 92 8 0 0 62211-6 2F 4 46 491 29 71 0 0 62211-6 2G 2 48 50 0 50 50 0 0 62211-6 2H 10 73 17 0 50 50 00 62212-9 2O 1 18 16 65 46 54 0 0 62212-9 2P 2 21 35 42 21 79 0 062212-9 2Q 2 24 17 57 37 63 0 0 62220-0 1F 3 93 4 0 0 100 0 0 62220-0 1G8 85 7 0 2 98 0 0 62224-2 2d 3 91 6 0 10 90 0 0 62224-2-2E 14 72 14 0 1585 0 0 62224-2 2F 3 94 3 0 0 100 0 0 62233-6 1d 6 71 23 0 25 75 0 062233-6 1F 2 95 3 0 29 71 0 0 62237-8 83 7 85 8 0 0 100 0 0 62237-8 8L 784 9 0 11 89 0 0 62261-1 2B 1 64 19 16 32 68 0 0 62262-4 3D 11 89 0 0 3862 0 0 62262-4 3E 12 82 6 0 86 14 0 0 62262-4 3F 11 89 0 0 62 38 0 062265-3 6C 0 9 7 84 69 31 0 0 62265-3 6D 0 10 3 87 2 98 0 0 62265-3 6E 217 3 78 5 95 0 0 62272-1 3G 0 32 11 57 7 93 0 0 62272-1 3P 0 28 5 67 919 0 0 62273-4 1G 7 87 6 0 4 96 0 0 62273-4 1H 4 93 3 0 73 27 0 0 62273-41I 8 83 9 0 3 97 0 0 62280-2 2H 8 86 6 0 9 91 0 0 62280-2 2L 6 90 4 0 397 0 0 62295-4 1B 3 85 12 0 45 55 0 0 62297-0 1C 17 63 20 0 12 88 0 0622970 1D 8 79 11 2 1 98 1 0 62298-3 1F 3 86 11 0 4 96 0 0 62298-3 1G 786 7 0 0 100 0 0 62300-5 6M 5 86 9 0 84 16 0 0 62300-5 6N 7 83 10 0 3859 3 0 62300-5 6O 6 81 13 0 1 79 20 0 62302-1 8H 0 58 4 38 28 72 0 062302-1 8I 0 52 3 45 13 85 2 0 62306-3 1E 4 79 17 0 19 81 0 0 62306-3 1F16 79 5 0 0 100 0 0 62315-7 4Q 35 63 2 0 51 49 0 0 62315-7 4R 38 62 0 087 13 0 0 62315-7 4S 27 73 0 0 95 5 0 0 62397-9 5K 0 43 0 57 45 48 7 062397-9 5Z 4 88 8 0 81 19 0 0

APPENDIX A List of Bacterial Artificial Chromosomes (BAC) and CentromereEnumeration Probes (CEP) Used for Fish Analysis

Gene name BAC ID Fluorescent tag ATM CTD2047A4 Spectrum Orange ™ ATRRP11-427D1 Spectrum Green ™ RP11-383G6 Spectrum Green ™ CHEK1RP11-712D22 Spectrum Orange ™ CCND1 RP11-699M19 Spectrum Aqua ™ H2AFXRP11-892K21 Spectrum Green ™ MRE11A RP11-685N10 Spectrum Orange ™ TP53RP11-199F11 Spectrum Orange ™

All BACs were purchased from the Children's Hospital Oakland ResearchInstitute (C.H.O.R.I.), Oakland, Calif. Two BACs RP11-427D1 andRP11-383G6 corresponding to the 5′ and 3′ ends of the ATR generespectively were used simultaneously.

APPENDIX B

Sequences for ATR and CHEK1 siRNA

ATR and CHEK1 siRNAs were obtained from Dharmacon Inc. The individualsequences from the smartpool are outlined below:

ATR sequences: (SEQ ID NO: 27) GAACAACACUGCTGGUUUG GAAGUCAUCUGUUCAUUAU(SEQ ID NO: 28) GAAAUAAGGUAGACUCAAU CAACAUAAAUCCAAGAAGA CHEK1 sequences:(SEQ ID NO: 29) UAAAGUACCACACAUCUUGUU UAUUGGAUAUUGCCUUUCUU(SEQID NO: 30) AUAUGAUCAGGACAUGUGGUU CCAUUGAUAGCCCAACUUCUU

APPENDIX C LIST OF ANTIBODIES USED FOR IMMUNOBLOTTING Concen- AntibodyType Company tration Total ATR Rabbit polyclonal Affinity 1:500-Bioreagents, 1:1000 Golden, CO. Total p53 (D-01) Mouse monoclonal SantaCruz 1:2000 Total ATM Rabbit polyclonal Cell Signaling 1:500 Total CHEK1Rabbit polyclonal Cell Signaling 1:1000 Total H2AX Mouse monoclonal CellSignaling 1:1000 Total MRE11A Goat polyclonal Santa Cruz 1:1000 pATM(Ser 1981) Rabbit polyclonal Cell Signaling 1:500 pATR (Ser 428) Mousemonoclonal Cell Signaling 1:500 pBRCA1 (Ser 1497) Goat polyclonal SantaCruz 1:1000 pCHEK1 (Ser 345) Rabbit polyclonal Cell Signaling 1:1000pCHK2 (Thr 68) Rabbit polyclonal Cell Signaling 1:1000 pSMC1 (Ser 987)Rabbit polyclonal Cell Signaling 1:1000 pCDC25C(Ser 216) Rabbitmonoclonal Cell Signaling 1:1000 γ-H2AX (Ser 139) Mouse monoclonalUpstate 1:1000 Actin Mouse monoclonal Sigma 1:2000 Tubulin Rabbitpolyclonal Santa Cruz 1:2000

APPENDIX D  Primer and probe sequences for QuMA and qRT-PCR analysesGene Forward (5′-3′) Reverse (5′-3′) Probe (5′-3′) MRE11AGGAATTAGTGAAATACCAGTTGGAA CTCTGAAACGACGTACCTCCTCA 6-FAM- (SEQ ID NO: 1)(SEQ ID NO: 4) TTCTTAAAGAACGTCATATTGATGCCCTCGA- TAMARA (SEQ ID NO. 7)E2AX CTCTGAAACGACGTACCTCCTCA CGCCCAGCAGCTTGTTG6-FAM-CACCGCTGAGATCCTGGAGCTGG- (SEQ ID NO: 2) (SEQ ID NO: 5) TAMARA(SEQ ID NO: 8) ATM GGCGGCAGTGCTGGAGTA TTTTAACTTGGTTTTATGACAATTGCT6-FAM-CTGCTCCTAATCCACCTCA (SEQ ID NO: 3) (SEQ ID NO: 6)TTTTCCATCGC-TAMARA (SEQ ID NO: 9) Microsatellite forward (5′-3′)Reverse (5′-3′) D11S4207 TAGAGATCCCGTTCGACTTG GCTGGGTGTTACACAGGAC(SEQ ID NO: 10) (SEQ ID NO: 11) D11S787 GTGGGCTTATTGTGGTAGTAGTCCAAGAGGAGGCAGGAGAGTC (SEQ ID NO: 12) (SEQ ID NO: 13) D11S1352TTTGTGAAATCTGAAGCACC TCCTTCATATCCTGAATCTCTG (SEQ ID NO: 14)(SEQ ID NO: 15) D11S901 CCCACATAGATTACTGGCCTC ATTCCTACATTAGCAGTTGGCA(SEQ ID NO: 16) (SEQ ID NO: 17) D11S1887 CTCCTCTGTATTCCCACAAAACACCTGACATTGTATCTAAACCTC (SEQ ID NO: 18) (SEQ ID NO: 19) D11S1358CTACAACCTGGATGAACCCTA AACCAACATTCTACTTTCTGTCT (SEQ ID NO: 20)(SEQ ID NO: 21) D11S917 ATGATGCCATATCTTGTCTTGA AATTTAAAGACAGATGCCAAGC(SEQ ID NO: 22) (SEQ ID NO: 23) D11S1893 CTAGTCCCTGGAACCTGGATGGCTGATGTGGGCTTTTTCAA (SEQ ID NO: 24) (SEQ ID NO: 25)

9. REFERENCES

-   Aaltonen, et al. (1993) Science, 260, 812-816.-   Abraham (2001) Genes Dev. 15(17), 2177-96.-   Åkervall, et al. (1997) Cancer, 79:3, 80-389-   Albertson, et al. (2003) Nat Genet, 34, 369-76.-   Alnemri, et al. (1996) Cell, 87(2), 171.-   Artandi, et al. (2000) Nature, 406(6796), 641-5.-   Ashman, et al. (2003) Br J Cancer, 89(5), 864-9.-   Austen, et all. (2007) J. Clin. One., 25(34), 5448-57.-   Bakkenist, et al. (2003) Nature, 421, 499-506.-   Balz, et al. (2003) Cancer Res., 63(6), 1188-91.-   Bao, et al. (2004) Oncogene. 23(33), 5586-93.-   Baocheng, et al. (2005) Cancer Res, 65(19), 8613-6.-   Bartek, et al. (2004) Nature Reviews Molecular Cell Biology, 5,    792-804.-   Bartek, et al. (2003) Cancer Cell, 3(5), 421-9.-   Bartkova, et al. (2005) Nature 434, 864-870.-   Bartkova, et al. (1995) Cancer Res, 55(4), 949-56.-   Bassing, et al. (2003) Cell, 114(3), 359-70.-   Bernard, et al. (1995) Genes Chromosomes Cancer, 13(2), 75-85.-   Bernstein, et al. (2002) Mutat Res., 511(2), 145-78.-   Bi, et al. (2004) Curr. Biol., 14, 1438-1353.-   Blackburn (1991) Nature, 350, 569-73.-   Bockmuhl, et al. (2000) Am J Pathol., 157(2), 369-75.-   Bockmuhl, et al. (2002) Genes Chromosomes Cancer., 33(1), 29-35-   Bockmuhl, et al. (1998) Head Neck., 20(2):145-51.-   Bodnar, et al. (1998) Science. 279(5349), 349-52.-   Boffetta, et al. (2006) Lancet Oncol. 7(2), 149-56.-   Bolt, et al. (2005) Oral Oncol., 41, 1013-1020.-   Broeks, et al. (2000) Am. J. Hum. Genet., 66, 494-500.-   Broker, et al. (2005) Clin Cancer Res., 11(9), 3155-62.-   Broustas, et al. (2004) J Biol Chem., 279(25), 26780-8.-   Brown, et al. (2003) Genes Dev., 17(5), 615-28.-   Brown (2005) Alcohol, 35, 161-168.-   Bryan, et al. (1997) Nature medicine, 3, 1271-74.-   Burma, et al. (2001) J Biol Chem., 276(45), 42462-7.-   Bursch, et al. (2000) Ann N Y Acad Sci., 926, 1-12.-   Busby, et al. (2000) Cancer Res., 60(8), 2108-12.-   Byun, et al. (2005) Genes Dev., 19(9), 1040-52.-   Cabral, et al. (2003) J Biol Chem., 278(20), 17792-9.-   Califano, et al. (1996) Cancer Res., 56(11), 2488-92.-   Califano, et al. (2000) Clinical Cancer Res, 6(2), 347-352.-   Carnero, et al. (2000) Nat Cell Biol., 2(3), 148-55.-   Casper, et al. (2002) Cell, 111(6), 779-89.-   Castedo, et al. (2004) Oncogene, 23(16), 2825-37.-   Celeste, et al. (2003) Cell, 114(3), 371-83.-   Chakrabarti, et al. (1991) Indian J Med Sci., 45(10), 273-5.-   Chang, et al. (1993) Am J Gastroenterol., 88(2), 174-86.-   Cheng, et al. (2005) Cancer Res., 65(19), 8646-54.-   Chin, et al. (1999) Cell, 97(4), 527-38-   Chu (1997) J Biol Chem., 272(39), 24097-100.-   Ciapponi, et al. (2004) Curr. Biol., 14, 1360-1366.-   Cohen (1997) Biochem J., 15, 326.-   Cole, et al. (1992) Science, 258(5088), 1650-4.-   Coquelle, et al. (1998) Mol Cell, 2(2), 259-65-   Coquelle, et al. (1997) Cell, 89(2), 215-25.-   Cortez, et al. (2001) Science, 294(5547), 1713-6.-   Croce, et al. (1999) J Clin Oncol., 17(5), 1618-24.-   Cuadrado, et al. (2006) J Exp Med., 203(2), 297-303.-   Dahiya, et al. (1997) Int. J. Cancer, 72, 283-288-   Dave, et al. (1992) Cancer, 70, 1017-23.-   de Boer, et al. (2000) Carcinogenesis, 21(3), 453-60.-   De Klein, et al. (2000) Curr Biol., 10(8), 479-82.-   De Villiers (1994) Curr Top Microbiol Immunol, 186, 1-12.-   Dickson, et al. (2000) Mol. Cell Biol., 20(4), 1436-1447.-   Dillehay, et al. (1988) NCI Monogr., (6), 173-6.-   Elbashir, et al. (2001) Nature, 411, 494-98.-   Enomoto, et al. (2004) Oncogene, 23(29), 5014-22.-   Eshleman, et al. (1996) Hum Mol Genet., 5 Spec No: 1489-94.-   Evans, et al. (2004) Head Neck, 26(1), 63-70.-   Evans, et al. (1998) Oncogene, 16, 2557-2264.-   Fabian, et al. (1996) J. Otolaryngol. 25, 88-93.-   Fedier, et al. (2003) Ann Oncol., 14, 938-945.-   Fesik (2005) Nat Rev Cancer, 5(11), 876-85.-   Forastiere, et al. (2001) N Engl J Med., 345(26), 1890-900.-   Fracchiolla, et al. (1997) Cancer, 79, 1114-1121.-   Smith, et al. (1999) Genes Dev, 13 916-934.-   Gatti, et al. (1991) Medicine (Baltimore), 70, 99-117.-   Gillison, et al. (2000) J Natl Cancer Inst., 92(9):709-20.-   Gisselsson (2003) Adv Cancer Res, 87, 1-29.-   Gisselsson, et al. (2002) Br J Cancer, 87(2), 202-7.-   Goldenberg, et al. (2004) Otolaryngol Head Neck Surg., 131(6),    986-93.-   Golding, et al. (2004) J. Biol. Chem., 279, 15402-15410.-   Gollin (2001) Head Neck, 23, 238-53.-   Gollin (2004) Curr Opin Oncol., 25-31.-   Gonzalez, et al. (2002) Laryngoscope, 112(3), 482-7.-   Gorgoulis, et al. (2005) Nature, 434(7035), 907-13.-   Graeber, et al. (1996) Nature, 379(6560), 88-91.-   Graves, et al. (2000) J Biol Chem., 275(8), 5600-5.-   Guan, et al. (2004) Cancer Res., 64(12), 4197-200.-   Guan, et al. (2001) Cancer Res., 61(9), 3806-9.-   Ha, et al. (2003) Crit Rev Oral Biol Med., 14(5), 363-9.-   Hammond, et al. (2003) J Biol Chem., 278(14), 12207-13.-   Hammond, et al. (2004) Cancer Res., 64(18), 6556-62.-   Hanahan, et al. (2000) Cell, 100(1), 57-70.-   Hefferin, et a (2005) DNA Repair (Amst)., 4(6), 639-48.-   Hekmat-Nejad, et al. (2000) Curr Biol., 10, 1565-1573.-   Helt, et al. (2005) J Biol Chem., 280(2), 1186-92.-   Hemmer, et al. (2006) Oncol Rep., 15(1), 243-6.-   Heselmeyer, et al. (1996) Proc Natl Acad Sci, 93, 479-484.-   Hickman, et al. (2002) Curr Opin Genet Dev., 12(1), 60-6.-   Ho, et al. (2004) Crit Rev Oral Biol Med, 15(4), 188-196.-   Hoeijmakers (1994) Eur J Cancer, 30A(13), 1912-21.-   Hoffelder, et al. (2004) Chromosoma, 112(8), 389-97.-   Holm (1982) Laryngoscope, 92(9 Pt 1), 1064-9.-   Huang, et al. (2006) Genes Chromosomes Cancer, 45, 1058-1069.-   Huang, et al. (2002) Proc Natl Acad Sci USA, 99(17), 11369-74.-   Huebner, et al. (2001) Nat Rev Cancer, 1(3), 214-21.-   Igney, et al. (2002) Nat Rev Cancer, 2(4), 277-88.-   Izzo, et al. (1998) Oncogene, 17(18), 2313-2322.-   Jackson, et al. (2000) Cancer Res., 60(3), 566-72.-   Jackson (2001) Biochem Soc Trans., 29(Pt 6), 655-61.-   Jackson (2002) Carcinogenesis, 23(5), 687-96.-   Janssen, et al. (2005) Head Neck, 27(7), 622-38.-   Jarvinen, et al. (2006) Oncogene PMID, 16715129.-   Jemal, et al. (2007) CA Cancer J. Clin., 57, 43-66.-   Jemal, et al. (2006) CA Cancer J Clin., 56(2), 106-30.-   Jin, et al. (2006) Cytogenet Genome Res., 115, 99-106.-   Jin, et al. (2002) Cancer Genet Cytogenet., 132, 85-96.-   Jin, et al. (2002) Int J Cancer, 98(3), 475-479.-   Jin, et al. (1998) Genes Chromosomes Cancer, 22(4), 312-20.-   Jiricny (2006) Nat Rev Mol Cell Biol., 7(5) 335-46.-   Jung, et al. (2006) Mol Cancer Res., 4(3), 169-76.-   Kalogeropoulos, et al. (2004) Cell Cycle, 3(9).-   Kang, et al. (2008) Mutat Res., 638(1-2), 17-25.-   Kao-Shan, et al. (1987) Cancer Res., 47(23), 6278-82.-   Kastan, et al. (2004) Nature, 432(7015), 316-23.-   Kawabe (2004) Mol Cancer Ther., 4, 513-9.-   Kaye, et al. (1993) Proc Natl Acad Sci USA, 90(19), 9150-4.-   Kettunen, et al. (2000) Cancer Genet Cytogenet., 117(1), 66-70-   Khanna, et al. (2001) Nat Genet., 27(3), 247-54.-   Kim, et al. (2000) Annu Rev Biochem., 69, 303-42.-   Kim, et al. (1994) Science, 266(5193), 2011-5.-   Kinzler, et al. (1996) Nature, 379(6560), 19-20.-   Kunkel, et al. (2005) Annu Rev Biochem., 74, 681-710.-   Lane (1992) Nature, 358(6381), 15-6.-   Lee, et al. (2004) Science, 304(5667), 93-6.-   Lengauer, et al. (1998) Nature, 396(6712), 643-9.-   Lese, et al. (1995) Genes Chromosomes Cancer, 12(4), 288-95.-   Levi, et al. (1998) Int J Cancer, 77(5), 705-9.-   Levine (1997) Cell, 88, 323-331.-   Li, et al. (1994) J Natl Cancer Inst., 86(20), 1524-9.-   Li, et al. (1998) Radiat Res., 149(4), 338-42.-   Lichter, et al. (1994) Methods Mol Biol, 29, 449-478.-   Liu, et al. (2000) Genes Dev., 14(12), 1448-59.-   Livingston, et al. (1990) Environ Mol Mutagen, 15(3), 136-44.-   Llewellyn, et al. (2004) Oral Oncol., 40(3), 304-13.-   Loeb, et al. (2003) Proc Natl Acad Sci USA, 100(3), 776-81.-   Lukas, et al. (2004) DNA Repair (Amst)., 3(8-9), 997-1007.-   Lundberg, et al. (1999) Eur J Cancer, 35, 531-539.-   Luo, et al. (2004) Mutat Res, 554(1-2), 375-85.-   Lyman (1992) Prim Care, 19(3), 465-79.-   Ma, et al. (2000) Oncogene, 19(23), 2739-44.-   Mack, et al. (2004) Radiat Res., 162(6), 623-34.-   Macleod (2000) Curr Opin Genet Dev., 10(1), 81-93.-   Majumder, et al. (2005) Cancer Epidemiol Biomarkers Prev., 14(9),    2106-12.-   Maniwa, et al. (2005) Cancer., 103(1), 126-32.-   Mao, et al. (1996) Nat Med., 2(6), 682-5.-   Martin-Granizo, et al. (1997) Otolaryngol Head Neck Surg., 117(3 Pt    1), 268-75.-   Maser, et al. (2002) Science, 297(5581), 565-9.-   Massague (2004) Nature, 432(7015), 298-306.-   Matsumoto, et al. (2004) Hum. Pathol., 35, 322-327.-   McClintock (1938) MO Agric Exp Sta Res Bull, 290, 1-48.-   McClintock (1939) Proc Natl Acad Sci USA, 25, 405-416.-   McKaig, et al. (1998) Head Neck, 20(3), 250-65.-   Meyn (1999) Clin Genet., 55(5), 289-304.-   Michalides, et al. (2002) Head Neck, 24(7), 694-704.-   Michalides, et al. (1997) Arch Otolaryngol Head Neck Surg., 123,    497-502.-   Michalides, et al. (1995) Cancer Res., 55, 975-978.-   Mineta, et al. (2000) Oral Oncol., 36, 194-198.-   Miyai, et al. (2004) Gynecol. Oncol., 94, 115-120.-   Mohindra, et al. (2002) Hum Mol Genet., 11(18), 2189-200.-   Momand, et al. (1998) Nucleic Acids Res., 26(15), 3453-9-   Monni, et al. (1998) Genes Chromosomes Cancer, 21(4), 298-307.-   Mork, et al. (2001) N Engl J Med., 344(15), 1125-31.-   Mumford, et al. (2006) Tob Control, 15(3), 166-71.-   Munger, et al. (1992) Cancer Surv., 12, 197-217.-   Musk, et al. (1990) Int J Radiat Biol., 57(6), 1105-12.-   Nagao, et al. (2000) Eur. J. Cancer, 368:466.-   Nakahara, et al. (2000) Cancer Lett, 160(1), 3-8.-   Neville. et al. (2002) CA Cancer J Clin., 52(4), 195-215.-   Nevins (2001) Hum Mol Genet., 10(7), 699-703.-   Nghiem, et al. (2001) Proc Natl Acad Sci USA, 98(16), 9092-7.-   Noguchi, et al. (2003) Oncol Rep. 10(5), 1393-400.-   Nugent, et al. (1998) Genes Dev., 12(8), 1073-85.-   O'Connell, et al. (2005) J Cell Sci., 118(Pt 1), 1-6.-   O'Driscoll (2003) Nat Genet., 33(4), 497-501.-   Ogden (2005) Alcohol, 35, 169-173.-   O'Hagan, et al. (2002) Cancer Cell, 2(2), 149-55.-   Okada, et al. (2004) Nat Rev Cancer, 4(8), 592-603.-   Parikh, et al. (2007) Genes Chromosomes Cancer, 46(8), 761-75.-   Parkin, et al. (2005) CA Cancer J Clin 55, 74-108.-   Parrilla-Castellar, et al. (2004) DNA Repair (Amst)., (8-9),    1009-14.-   Paull, et al. (2000) Curr. Biol., 10, 886-895.-   Pei, et al. (2001) Genes Chromosomes Cancer. 31(3), 282-7.-   Peltomaki (2003) J Clin Oncol., 21(6), 1174-9.-   Petti (2003) Oral Oncol., 39(8), 770-80.-   Phelps, et al. (1998) Antivir Chem Chemother., 9(5), 359-77.-   Poppe. et al. (2004) Blood, 103(1), 229-35.-   Poschl, et al. (2004) Alcohol Alcohol, 39(3), 155-65.-   Poschl, et al. (2004) Proc Nutr Soc., 63(1), 65-71.-   Prime, et al. (2001) Oral Oncol., 37(1), 1-16.-   Quintyne, et al. (2005) Science, 307(5706), 127-9.-   Ragin, et al. (2004) Int J Cancer, 110(5), 701-9.-   Redon, et al. (2002) Cancer Res., 62(21), 6211-7.-   Redon, et al. (2001) Cancer Res., 61(10), 4122-9.-   Reichart (2001) Clin Oral Investig, 5(4), 207-13.-   Reshmi, et al. (2007) Cytogenet Genome Res., 116, 46-52.-   Reshmi, et al. (2005) J Dent Res, 84(2), 107-17.-   Rhodus (2005) Dent Clin North Am., 49(1), 143-65.-   Riedl, et al. (2004) Nat Rev Mol Cell Biol., 5(11), 897-907.-   Rogakou, et al. (2000) Biol. Chem., 275, 9390-9395.-   Rogakou, et al. (1998) J. Biol. Chem. 273, 5858-5868.-   Roh, et al. (2000) Cancer Res. 60, 6496-6502.-   Sabatier, et al. (2005) Mol Cancer Res., 3(3), 139-50.-   Sanchez, et al. (1997) Science, 277(5331), 1497-501.-   Sarbia, et al. (2007) British Journal of Cancer, 97(10), 1404-8.-   Sarkaria, et al. (1999) Cancer Res., 59(17), 4375-82.-   Sartor, et al. (1999) Br J Cancer, 80(1-2), 79-86.-   Sartor, et al. (1999) Br J Cancer, 80(1-2), 79-86.-   Sattler, et al. (1999) Prostate, 39(2), 79-86.-   Saunders, et al. (2000) Proc Natl Acad Sci USA, 97, 303-308.-   Schantz, et al. (2002) Arch Otolaryngol Head Neck Surg., 128(3),    268-74.-   Schraml, et al. (1999) Clin Cancer Res., 5, 1966-1975.-   Schuuring, et al. (1998) Cell Adhes Commun., 6, 185-209.-   Schuuring, et al. (1992) Oncogene, 7(2), 355-361.-   Schuuring (1995) Gene, 159(1), 83-96.-   Seitz, et al. (1998) Recent Dev Alcohol, 14:67-95.-   Seitz, et al. (2002) Nutritional Toxicology, 2nd edn, 122-154.-   Serrano, et al. (1996) Cell, 85(1), 27-37.-   Shao, et al. (1997) Cancer Res., 57(18), 4029-35.-   Sherr, et al. (2002) Cancer Cell, 2(2):103-12.-   Sherr (1998) Genes Dev., 12(19), 2984-91.-   Shiloh (2001a) Curr Opin Genet Dev, 11(1):71-7.-   Shiloh (2001b) Nat Rev Cancer, 3(3):155-68.-   Shiloh, (2003) Cell Cycle, 2(2), 116-7.-   Shintani, et al. (2001) Oral Oncol., 37(6), 498-504.-   Shiu, et al. (2004) Eur J Cancer Prev., 13(1), 39-45.-   Shuster, et al. (2000) Genes Chromosomes Cancer, 28(2), 153-63.-   Sidransky (1995) Curr Opin Oncol, 7(3), 229-33.-   Silverman (2001) J Am Dent Assoc., 132:7 S-11S.-   Singh, et al. (2002) Am J Pathol., 161(2), 365-71.-   Siprashvili, et al. (1997) Proc Natl Acad Sci USA, 94(25), 13771-6.-   Smith, et al. (1999) Genes Dev., 13(8), 916-34.-   Smith, et al. (1998) Nat Genet, 19(1), 39-46.-   Soderlund, et al. (2007) Int. J. Rad. Onco., Bio., Phys., 68(1),    50-8.-   Spitkovsky, et al. (2002) J Biol Chem., 277(28), 25576-82.-   Stein, et al. (2002) Genes Chromosomes Cancer, 34(3), 333-40.-   Stell (1991) Head Neck, 13(4), 277-81.-   Stewart, et al. (1999) Cell, 99, 577-587.-   Stich, et al. (1992) Int J Cancer, 50(2), 172-6.-   Sticht, et al. (2005) Br J Cancer, 92(4), 770-4.-   Stoltzfus, et al. (2005) Int J Gynecol Cancer, 15(1), 120-6.-   Storz (2005) Front Biosci., 10, 1881-96.-   Stracker, et al. (2004) DNA Repair(Amst)., 3(8-9), 845-54.-   Sudbo, et al. (2002) JCO, 20, 456-462.-   Sweasy, et al. (2006) Cell Cycle, 5(3), 250-9.-   Syljuasen, et al. (2005) Mol Cell Biol., 25(9), 3553-62.-   Takai, et al. (2000) Genes Dev., 14(12), 1439-47.-   Telmer, et al. (2003) Hum Mutat., 2, 158-65.-   Thelen, et al. (1999) Cell. 96(6), 769-70.-   Theunissen, et al. (2003) Mol Cell, 12(6), 1511-23.-   Tibbetts, et al. (1999) Genes Dev., 13(2), 152-7.-   Tomar (2003) Am J Med Sci., 326(4), 248-54.-   Tomlinson, et al. (1995) J. Clin. Pathol., 48, 424-428.-   Trujillo, et al. (1998) J Biol Chem., 273(34), 21447-50.-   Tsuchiya, et al. (1998) Anticancer Res, 18(1B), 657-66.-   Tsuchiya, et al. (1998) Anticancer Res., 18(1B), 657-66.-   Van Dyke, et al. (1994) Genes Chromosomes Cancer, 9, 192-206.-   Vaupel, et al. (2001) Semin Oncol., 28(2 Suppl 8), 29-35.-   Vaziri, et al. (1998) Curr Biol., 8(5), 279-82.-   Virgilio, et al. (1996) Proc Natl Acad Sci USA, 93(18), 9770-5.-   Voorhoeve, et al. (2003) Cancer Cell, 4(4), 311-9.-   Wall, et al. (2003) Lancet, 5; 362(9393), 1401-3.-   Wang, et al. (2004) Cancer Res., 64, 64-71.-   Wang, et al, (2003) J Biol Chem., 278(33), 30869-74.-   Ward, et al. (2001) J Biol Chem., 276(51), 47759-62.-   Weinberg (1995) Cell, 5; 81(3):323-30.-   Werness, et al. (1990) Science, 248(4951), 76-9.-   Westphal, et al. (1998) Cancer Res., 58, 5637-5639.-   White, et al. (2007) Oral Onco., 43(7), 701-12/-   Wood, et al. (2001) Science, 291(5507), 1284-89.-   Wood (1997) J Biol Chem, 272(38), 23465-8.-   Xu, et al. (2001) Mol Cell Biol, 21(10), 3445-50.-   Yabro (1992) Semin Oncol Nurs., 8(1), 30-9.-   Yancik (2005) Cancer J, 11(6), 437-41.-   Yang, et al. (2001) Cancer Genet Cytogenet, 131(1), 48-53.-   Ye, et al. (2007) Cancer, 109(9), 1729-35.-   Yeh, et al. (2003) Oncol Rep, 10(3), 659-63.-   Young, et al. (2004) Nat Rev Cancer, 4(10), 757-68.-   Yu, et al. (2004) Curr Opin Oncol., 16(1), 19-24.-   Zatkova, et al. (2004) Genes Chromosomes Cancer, 39(4), 263-76.-   Zhao, et al. (2002) J Biol Chem., 277(48), 46609-15.-   Zhivotovsky, et al. (2004) Nat Rev Mol Cell Biol, 5(9), 752-62.-   Zhou, et al. (2003) Cancer Biol Ther., 2(4 Suppl 1), S16-22.-   Zhou, et al. (2003) Prog Cell Cycle Res, 5, 413-21.-   Zou, et al. (2003). Science, 300(5625), 1542-8.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

We claim:
 1. A method of identifying a patient suffering from a cancerwhich is likely to be refractory to therapy comprising (i) identifyingan 11q deletion in a patient sample considered to be representative ofthe cancer and, (ii) identifying, in the patient sample, overexpressionof a gene selected from the group consisting of ATR, CHEK1, or acombination thereof, wherein an 11q deletion and overexpression of ATR,CHEK1, or both, indicates that the cancer is likely to be refractory tothe therapy.
 2. The method of claim 1 where the patient is sufferingfrom a cancer selected from the group consisting of squamous cellcarcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, multiplemyeloma, and prostate cancer.
 3. The method of claim 2 where the patientis suffering from an oral squamous cell carcinoma.
 4. The method ofclaim 1, wherein overexpression of ATR is identified.
 5. The method ofclaim 1, wherein overexpression of CHEK1 is identified.
 6. A method oftreating a patient suffering from a cancer, comprising (i) identifyingthe patient as being likely to benefit from inhibition of the ATR/CHEK1pathway by a method comprising (i) (a) identifying an 11q deletion in apatient sample considered to be representative of the cancer, and/or (b)identifying, in the patient sample, overexpression of a gene selectedfrom the group consisting of ATR, CHEK1, CCND1, or a combinationthereof, wherein an 11q deletion and overexpression of ATR, CHEK1,CCND1, or a combination thereof, indicates that the cancer is likely tobenefit from inhibition of the ATR/CHEK1 pathway; and (ii) treating thepatient with an inhibitor of the ATR/CHEK1 pathway.
 7. The method ofclaim 6 where the patient is suffering from a cancer selected from thegroup consisting of squamous cell carcinoma, breast carcinoma, ovariancarcinoma, lung carcinoma, multiple myeloma, and prostate cancer.
 8. Themethod of claim 7 where the patient is suffering from an oral squamouscell carcinoma.
 9. The method of claim 6, wherein overexpression of ATRis identified.
 10. The method of claim 6, wherein overexpression ofCHEK1 is identified.
 11. A method of identifying a patient sufferingfrom a cancer which is likely to be refractory to therapy and/or whichis likely to benefit from inhibition of the ATR/CHEK1 pathway,comprising (i) exposing cells in a patient sample considered to berepresentative of the cancer to a source of free-radicals such asionizing radiation; and (ii) determining whether there is a higherpercentage of cells in the S and G2M phases relative to control cells(representative of normal tissue); wherein a higher percentage of cellsin the S and G2M phases indicates that the cancer is likely to berefractory to therapy and/or is likely to benefit from inhibition of theATR/CHEK1 pathway.
 12. The method of claim 11 where the patient issuffering from a cancer selected from the group consisting of squamouscell carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma,multiple myeloma, and prostate cancer.
 13. The method of claim 12 wherethe patient is suffering from an oral squamous cell carcinoma.
 14. Amethod of treating a patient in need of such treatment comprisingadministering, to the patient, (i) a composition comprising an effectiveamount of a compound selected from the group consisting of a smallinterfering RNA, an antisense RNA, and a catalytic RNA, in an amounteffective in inhibiting expression of a gene selected from ATR andCHEK1, and (ii) a DNA-damaging agent.
 15. The method of claim 14 wherethe patient is suffering from a cancer selected from the groupconsisting of squamous cell carcinoma, breast carcinoma, ovariancarcinoma, lung carcinoma, multiple myeloma, and prostate cancer. 16.The method of claim 15 where the patient is suffering from an oralsquamous cell carcinoma.
 17. A method of treating a patient in need ofsuch treatment comprising administering to the patient (i) a compositioncomprising an effective amount of a small molecule inhibitor of theATR/CHEK1 pathway and (ii) a DNA-damaging agent.
 18. The method of claim17 where the patient is suffering from a cancer selected from the groupconsisting of squamous cell carcinoma, breast carcinoma, ovariancarcinoma, lung carcinoma, multiple myeloma, and prostate cancer. 19.The method of claim 18 where the patient is suffering from an oralsquamous cell carcinoma.
 20. A kit comprising one more nucleic acidprobe that may be used to identify a deletion in 11q and one or morenucleic acid probe which may be used to detect overexpression of a geneselected from the group consisting of ATR and CHEK1.
 21. The kit ofclaim 20 further comprising an antibody directed against a proteinselected from the group consisting of ATR, CHEK1, MRE11A, CCND1, H2AFX,p53, p21 and CHEK1.
 22. The kit of claim 21 where one or more nucleicacid probe is detectably labeled.
 23. The kit of claim 20 where one ormore antibody is directly or indirectly detectably labeled.
 24. A methodof identifying a patient suffering from a cancer which is likely to berefractory to therapy comprising (i) identifying an 11q deletion in apatient sample considered to be representative of the cancer; (ii)identifying, in the patient sample, overexpression of a gene selectedfrom the group consisting of ATR, CHEK1, CCND1, or a combinationthereof; and (iii) identifying, in the patient sample, a decrease orabsence of p53 expression and/or activity, wherein an 11q deletion,overexpression of ATR, CHEK1, CCND1, or a combination thereof, and adecrease or absence of p53 expression and/or activity indicates that thecancer is likely to be refractory to the therapy and may benefit frominhibition of the ATR/CHEK1 pathway.
 25. The method of claim 24 wherethe patient is suffering from a cancer selected from the groupconsisting of squamous cell carcinoma, breast carcinoma, ovariancarcinoma, lung carcinoma, multiple myeloma, and prostate cancer.
 26. Amethod of treating a patient suffering from a cancer, comprising (i)identifying the patient as being likely to benefit from inhibition ofthe ATR/CHEK1 pathway by a method comprising (a) identifying an 11qdeletion in a patient sample considered to be representative of thecancer and/or (b) identifying, in the patient sample, overexpression ofa gene selected from the group consisting of ATR, CHEK1, CCND1, or acombination thereof, and (c) identifying, in the patient sample, adecrease or absence in p53 expression and/or activity, wherein an 11qdeletion and overexpression of ATR, CHEK1, CCND1, or a combinationthereof and/or a decrease or absence in p53 expression and/or activityindicates that the cancer is likely to benefit from inhibition of theATR/CHEK1 pathway; and (ii) treating the patient with an inhibitor ofthe ATR/CHEK1 pathway.